From owner-proteins@net.bio.net Fri Jul 01 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!gatech!newsxfer.itd.umich.edu!zip.eecs.umich.edu!umn.edu!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!ipp-garching.mpg.de!nmrvex.biochem.mpg.de!ross From: ross@nmrvex.biochem.mpg.de (Alfred Ross) Newsgroups: bionet.molbio.proteins Subject: MIMETICS Review Date: 2 Jul 1994 19:14:21 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 10 Message-ID: <2v4eadINN1kag@sat.ipp-garching.mpg.de> NNTP-Posting-Host: nmrvex.biochem.mpg.de X-Newsreader: TIN [version 1.1 PL8] Hi Colleagues, does anybody know a good review article about peptide mimetics which a phycicist can read to get an overlook about the subject. Thanks: Alfred Ross From owner-proteins@net.bio.net Fri Jul 01 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!darwin.sura.net!guvax.acc.georgetown.edu!oneillp From: oneillp@guvax.acc.georgetown.edu (Patricia O'Neill-Brown) Newsgroups: bionet.molbio.proteins Subject: 4th Annual JICST/NTIS Conference Message-ID: <1994Jul2.152049.11038@guvax> Date: 2 Jul 94 15:20:49 -0500 Distribution: world Organization: Georgetown University Lines: 380 FOURTH JICST/NTIS CONFERENCE Boston July 14-15, 1994 Learn the Latest Methods in Locating and Acquiring Japanese Scientific and Technical Information The conference is the fourth in a highly successful series to be organized jointly by the U.S. Department of Commerce National Technical Information Service (NTIS) and the Japan Information Center of Science and Technology (JICST). It is designed to provide practical assistance for those who need access to Japanese scientific and technical information (JSTI). The conference will address the basic questions of what STI is available from Japan and how American users can gain access. The conference will draw heavily on the experiences of U.S. companies, academic institutions and Government agencies. In addition, the major suppliers of JSTI will be on hand in an exhibit hall to discuss and demonstrate their products and services. AGENDA Thursday, July 14, 1994 7:30-8:30 am Registration/Coffee 8:30-9:00 am Welcome * Dr. Donald Johnson, Director, NTIS * Mr. Tateo Armimoto, Director, S&T Information Division, Science and Technology Agency, Japan 9:00-9:45 am Keynote Addresses * Mr. Sumio Horiuchi, Vice-President, JICST * Dr. Mary Good, Under Secretary for Technology U.S. Department of Commerce, Washington DC 9:45-10:00 am Refreshment Break 10:00-11:30 am Panel: How Small and Medium-Sized Companies Acquire and use Japanese Science and Technology Information (JSTI) (Case studies on how companies have successfully met their JSTI needs. Why they perceive access to JSTI as desirable. Pitfalls to be avoided. The most effective approaches. How the acquired material is used in the company.) Moderator: Dr. Russell Jamison, Vice President for R&D, Smith & Nephew Richards Inc. Speakers: Michael W. Chinworth, TASC/Rosslyn, Arlington, Virginia Topic: Technology Assessment: Challenges to Small and Medium-Sized Companies. Lee Howell, Research Assistant and External Affairs Officer for Japan, Center for Strategic and International Studies, Washington, DC. Topic: Understanding Japan's Science and Technology Administration and Policy Regime. Stephen Blakely, President, Blakely and Associates, Alton, Illinois Topic: The Relevance of Japanese Scientific and Technical Information to Technology-Based Business Studies. 11:30-12:00 Demonstration: First public demonstration of MIT CD/ROM-based interactive Japanese language/culture system *Professor Shigeru Miagawa, Director, Japanese Language and Culture Program, MIT 12:00-1:30 pm Luncheon and demonstrations in the exhibit area 1:30-3:00 pm Panel: Commercial Packaging and Dissemination of JSTI (Providing access to JSTI on a commercial basis. What is available in the marketplace? How has the market responded?) Moderator: David Andrews, Chief Executive Officer, InterLingua, Los Angeles, California Speakers: Hilary Handwerger, National Center for Manufacturing Sciences, Ann Arbor, Michigan Hal Morimoto, Executive Director, Comline Business Data Inc., New York City Madeline Dovale, SEMATECH Corporation, Austin, Texas Hitoshi Inoue, National Academic Center for Science Information System, Tokyo Topic: NACSIS International: 1994 Update Ryo Sasaki, National Diet Library, Tokyo Topic: Acquisition of JSTI in the National Diet Library 3:00-3:15 pm Refreshment Break 3:15-4:30 pm Panel: Acquiring and Understanding Japanese Patent Information (How to identify and obtain Japanese patent information. How Japanese patent practice differs from American practice. Obtaining permission to translate and reprint copyrighted Japanese materials.) Moderator: Mindy L. Kotler, President, Japan Information Access Project Speakers: Gary Hamilton, Esq., Gambrell, Wilson & Hamilton, Austin, Texas Topic: Comparing the Japanese and U.S. Patents Systems. Steven W. Johnston, Patent Translator, Seabrook, Texas Topic: Using Japanese Online Patent Information. Jeffrey Foreman, Esq. Patent Attorney, IBM Corporation, Arlington, Virginia Topic: Using and Finding Japanese Patent Information. 4:45-5:15 pm Demonstration: New MIT Japanese database gateway system * Carol Fleishauer, Associate Director for Collection Services, MIT Library System 5:15-5:30 pm Refreshment Break 5:30-7:00 pm Panel: Using Databases to Obtain JSTI (Part 1) (What databases are available and what are their characteristics? Actual user experiences. How can the databases better meet the needs of American users? What does the future hold?) Moderator: Maureen H. Donovan, Japanese Studies Librarian, The Ohio State University Speakers: Glenn Hoetker, NASA Scientific and Technical Information Program and RMS, Inc., Washington, DC Yukio Sueyoshi, Nihon Keizai Shimbun America, Inc. Mari Ikeda, Japan Center for Information and Cultural Affairs Topic: Japan Information Center Network. Koji Tamura, JICST, Tokyo Topic: JICST's Company Information File. Friday, July 15, 1994 8:00-8:30 am Coffee 8:30-9:00 am Special Address Dr. Daniel Wang, Chevron Professor and Director of the Biotechnology Process Engineering Center, MIT Topic: Access to Japanese Biotechnology. 9:00-10:30 am Panel: Using Databases to Obtain JSTI (Part 2) (What databases are available and what are their characteristics? Actual user experiences. How can the databases better meet the needs of American users? What does the future hold?) Moderator: Maureen H. Donovan, Japanese Studies Librarian, The Ohio State University Speakers: Alan Engel, International Science and Technology Associates, Inc. Keisuke Okuzumi, Japan Database Promotion Center, Tokyo Topic: Summary of Japan's Data Service. Akira Ohashi, Electronic Devices Information Service Co., Ltd. Topic: ELNET and ELISNET. Shereen Hubbard, Foreign Broadcast Information Service, Washington, DC 10:30-10:45 am Refreshment break 10:45-12:15 pm Panel: Tools for Understanding and Handling JSTI (Translation services. Machine-aided translation. Computer processing of Japanese language text. Dissemination and exchange through network services.) Moderator: Carl Kay, President, Japanese Language Services, Inc., Boston, Massachusetts Speakers: Carl W. Hoffman, President, Basis Technology Corporation, Boston, Massachusetts Topic: Reading Japanese Text Online. Glenn Akers, Ph.D, President, Language Engineering Corporation, Belmont, Massachusetts Topic: Machine Translation. Burton Lee, X-Guide Program Manager, U.S.-Japan Technology Center, Stanford University, Stanford, California. Topic: Using the Internet to Access JSTI. Carl Kay (affiliation above) Topic: Translating Japanese STI. Yoshiko Shirakizawa, JICST Topic: Current Status and Future of the JICST Machine Aided Translations System. 12:15-1:45 pm Luncheon and demonstrations in the exhibit area 1:45-2:15 pm Special Address: Michiyuki Uyenohara, Special Consultant, NEC Corporation Topic: Science and Technology Strategies of Japanese Corporations. 2:15-3:45 pm Panel: Advances in Teaching and Learning Technical Japanese (University programs, including internship programs; target audience; new teaching methods and aids, including those involving computer/network technologies) Moderator: Dr. Michio Tsutsui, Director, Technical Japanese Program, College of Engineering, University of Washington, Seattle, Washington Speakers: Richard Dasher, Associate Director, U.S.-Japan Technology Management Center, Stanford University, Stanford, California Topic: Technical Japanese Language and Cross-Cultural Training at Stanford University. Dr. James L. Davis, Assistant Professor (Technical Japanese), Department of Engineering Professional Development, University of Wisconsin-Madison, Wisconsin Topic: Computer-Assisted Distance Education and Technical Japanese Instruction at the University of Wisconsin-Madison. (Prof. Davis will speak by telephone from Tokyo. He will be assisted by Mr. Wayne Utke of the University of Wisconsin.) Dr. David Mills, Associate Professor, Department of East Asian Languages and Literatures, University of Pittsburgh, Pennsylvania and Director, Technical Japanese Language Project, Massachusetts Institute of Technology, Boston Topic: How to Make an Engineer Functional in Japanese: Innovations at MIT. Dr. Michio Tsutsui (affiliation above) Topic: Two Programs for Engineers and Scientists at the University of Washington. 3:45-4:00 pm Closing Remarks *Sumio Horiuchi, JICST Vice President 4:00 pm Adjournment REGISTRATION Registration Fee: $295 (Cancellation is subject to $50 fee). You can register for the NTIS/JICST Conference by contacting Barbara Payne at (703) 487-4819. (National Technical Information Service, 5285 Port Royal Road (306 F) Springfield, VA 22161). Quote Order Number: CONF94-1. HOTEL RESERVATIONS The conference will be held at the: Copley Plaza Hotel 138 James Avenue Boston, MA 02116 Reservations: (617) 267-5300 A special conference rate of $129/night (single or double) is being offered. You must mention the NTIS/JICST Conference to receive this special rate. For hotel reservations, contact the hotel directly. Rooms held open until June 13.  From owner-proteins@net.bio.net Sun Jul 03 23:00:00 1994 Path: biosci!UV4.INVEST.UV.MX!evargas From: evargas@UV4.INVEST.UV.MX (Enrique Vargas Madrazo) Newsgroups: bionet.molbio.proteins Subject: Prof. K. Kleinkauf e-mail Date: 4 Jul 1994 15:18:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 31 Sender: daemon@net.bio.net Distribution: world Message-ID: <9407042215.AA00982@uv4.invest.uv.mx> NNTP-Posting-Host: net.bio.net ---------- X-Sun-Data-Type: text X-Sun-Data-Description: text X-Sun-Data-Name: text X-Sun-Charset: us-ascii X-Sun-Content-Lines: 2 Dear Colleagues: Anybody knows the Prof. K. Kleinkauf e-mail? I can't find it, Your help will be greatly appreciated. ---------- X-Sun-Data-Type: default X-Sun-Data-Name: logo.evm X-Sun-Content-Lines: 16 X-Sun-Charset: us-ascii ****************************************************************************** * * evargas@uv4.invest.uv.mx * * Dr. Enrique Vargas-Madrazo * evargas@speedy.coacade.uv.mx * * * optional: salazar@redvax1.dgsca.unam.mx * ****************************************************************************** * Laboratorio de Biologia Molecular * Phone number: (28)125757 * * e Inmunologia Teorica. * FAX number: (28)125757 * ****************************************************************************** * Instituto de Investigaciones Biologicas * Universidad Veracruzana * * * Xalapa, Veracruz; Mexico. * ****************************************************************************** * POSTAL ADRESS: * * Juan de la Barrera 54, * * Col. Electricistas, C.P. 91000 * * Xalapa, Veracruz; Mexico * ************************************** From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!post.its.mcw.edu!news.doit.wisc.edu!f181-216.net.wisc.edu!kenp From: Ken Prehoda Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 5 Jul 1994 14:25:43 GMT Organization: Univ of Wisc-Madison Lines: 45 Distribution: world Message-ID: <2vbqh7$6e8@news.doit.wisc.edu> References: <2vbn6u$j4e@usenet.rpi.edu> NNTP-Posting-Host: f181-216.net.wisc.edu X-Newsreader: Nuntius Version 1.2 X-XXMessage-ID: X-XXDate: Tue, 5 Jul 1994 15:22:12 GMT In article <2vbn6u$j4e@usenet.rpi.edu> Mark J. Dresser, writes: >QUESTIONS: What role do/could tertiary interactions play in stabilizing >the alpha-helix? Probably a significant role, but this is only my opinion. There is a great deal of work being done on small, helix forming peptides (cf. Baldwin and coworkers). But, the question one must ask is whether secondary structure is present in the unfolded protein. If it is not (which seems to be the case - molten globules and other curiosities not withstanding), then this seems good evidence that tertiary structure is indeed necessary for secondary structure formation. >Does a protein first fold into its secondary structure elements and then into >its tertiary structure, afterwhich the original secondary structure elements >remain as before the folding into the tertiary structure? (If yes or no, could >you please provide references). Since protein folding is believed to be thermodynamically controlled (OK, see Agard, et al. for counterviews), this is really irrelevant to what you seem to be getting at. Remember that G is a state function and is therefore independent of path. >Rose proposes that "the folded structure of a protein is encrypted in its >aa secquence, written in a *code* that remians obscure". (Rose, G.D. et >al. (1994) _Science_ 264, 1126). What is the status of this obscure *code* >that he is referring to? Is the existence of this *code* a widely held >belief? The status of this "code" is abysmal. We know very little about the relationship of primary and higher order structures of proteins. The existence of such a code is certainly widely held (cf. Anfinsen and coworkers). If we believe that all of the information is contained in the sequence itself, then it follows that there should be some type of code that determines protein structure. But maybe the code is different for every protein? Looking forward to further discussion, Ken Prehoda kenp@nmrfam.wisc.edu From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!howland.reston.ans.net!EU.net!sunic!news.lth.se!news.lu.se!q700.plantbio.lu.se!Lars.S From: RoundUp Subject: Visiblot AP from USB any users? Message-ID: <1994Jul5.081744.18901@nomina.lu.se> X-Xxmessage-Id: X-Xxdate: Tue, 5 Jul 94 08:12:51 GMT Sender: news@nomina.lu.se (USENET News System) Nntp-Posting-Host: q700.plantbio.lu.se Organization: Plant biochemistry U of Lund X-Useragent: Version 1.1.3 Date: Tue, 5 Jul 1994 08:17:44 GMT Lines: 9 Has anyone used the visiblot AP reagent from USB on western blotts? Does it work as good as they state? Is the sensitivity as good as with the BCIP/NBT system? All comment appreciated! _________________________________________________________ Lars Snogerup Lars.S@Plantbio.lu.se Plant Biochemistry Lund University Sweden From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!cs.utexas.edu!howland.reston.ans.net!EU.net!sunic!news.lth.se!news.lu.se!q700.plantbio.lu.se!Lars.S From: RoundUp Subject: Visiblot AP from USB any users? Message-ID: <1994Jul5.081955.19071@nomina.lu.se> X-Xxmessage-Id: X-Xxdate: Tue, 5 Jul 94 08:14:05 GMT Sender: news@nomina.lu.se (USENET News System) Nntp-Posting-Host: q700.plantbio.lu.se Organization: Plant biochemistry U of Lund X-Useragent: Version 1.1.3 Date: Tue, 5 Jul 1994 08:19:55 GMT Lines: 9 Has anyone used the visiblot AP reagent from USB on western blotts? Does it work as good as they state? Is the sensitivity as good as with the BCIP/NBT system? All comment appreciated! _________________________________________________________ Lars Snogerup Lars.S@Plantbio.lu.se Plant Biochemistry Lund University Sweden From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!news.cac.psu.edu!news.tc.cornell.edu!travelers.mail.cornell.edu!newsstand.cit.cornell.edu!news.graphics.cornell.edu!ghost.dsi.unimi.it!news.unige.it!news.ibf.unige.it!moana.ibf.unige.it!biodev From: biodev@moana.ibf.unige.it (Gruppo Biodevices) Newsgroups: bionet.molbio.proteins Subject: EC 1.10.3.2 Date: 5 Jul 1994 13:59:31 GMT Organization: String to put in the Organization Header Lines: 16 Message-ID: <2vbp03$j8@barbarella.ibf.unige.it> NNTP-Posting-Host: biodev@moana.ibf.unige.it Keywords: info on structure X-Newsreader: TIN [version 1.2 PL2] Hi to everybody, I'm searching for the structure of LACCASE on PDB : do you know if the structure of this protein has been yet resolved? if yes, please send me the name of the file on pdb or other information regarding the number of Lys residues on the surface of this protein. thank you my address is: Marco Lanzi Inst.of Biophysics School of Medicine-Genova Italy e-mail: biodev@ibf.unige.it From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!sdd.hp.com!nigel.msen.com!emory!swrinde!pipex!lyra.csx.cam.ac.uk!bjd12 From: bjd12@cus.cam.ac.uk (Ben Davis) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 5 Jul 1994 15:24:53 GMT Organization: University of Cambridge, England Lines: 68 Distribution: world Message-ID: <2vbu05$det@lyra.csx.cam.ac.uk> References: <2vbn6u$j4e@usenet.rpi.edu> <2vbqh7$6e8@news.doit.wisc.edu> NNTP-Posting-Host: grus.cus.cam.ac.uk X-Newsreader: TIN [version 1.2 PL2] Ken Prehoda (kenp@nmrfam.wisc.edu) wrote: : In article <2vbn6u$j4e@usenet.rpi.edu> Mark J. Dresser, writes: : >QUESTIONS: What role do/could tertiary interactions play in stabilizing : >the alpha-helix? : Probably a significant role, but this is only my opinion. There is a : great deal of work being done on small, helix forming peptides (cf. : Baldwin and coworkers). But, the question one must ask is whether : secondary structure is present in the unfolded protein. If it : is not (which seems to be the case - molten globules and other : curiosities not withstanding), then this seems good evidence that : tertiary structure is indeed necessary for secondary structure : formation. I think that more proteins need to be characterised under denaturing conditions before the question of structure in unfolded proteins can be answered properly. Certainly in the cases where unfolded proteins have been characterised (434 repressor, FK506 BP) structures have been found. They tend to be marginal, rather than persistent, but they do seem to be formed. This kind of implies that local interactions can form, and so presumably would form early in a folding reaction. : >Rose proposes that "the folded structure of a protein is encrypted in : its : >aa secquence, written in a *code* that remians obscure". (Rose, G.D. et : >al. (1994) _Science_ 264, 1126). What is the status of this obscure : *code* : >that he is referring to? Is the existence of this *code* a widely held : >belief? : The status of this "code" is abysmal. We know very little about the : relationship of primary and higher order structures of proteins. The : existence of such a code is certainly widely held (cf. Anfinsen and : coworkers). If we believe that all of the information is contained : in the sequence itself, then it follows that there should be some : type of code that determines protein structure. But maybe the code : is different for every protein? I think that its a fairly safe bet that the structure of a protein is determined by its sequence, and that its unlikely (and overly pessimistic) to think that the rules governing this final structure are different for each sequence. However, it does seem that its complicated, and that there's no easy answers to the question of the folding pathway, or even the final fold. I personally suspect that the solution will involve looking for adoption of structures in a hierachical manner - clusters, local secondary structures, tertiary structures, and so on. : Looking forward to further discussion, Indeed. Could be a good thread. : Ken Prehoda : kenp@nmrfam.wisc.edu Ben -- ______________________________________________________________________________ Ben Davis, MRC Protein Function and Design, Cambridge, UK ______________________________________________________________________________ "They can make me do it, but they can't make me do it with dignity." From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!galaxy.ucr.edu!library.ucla.edu!ihnp4.ucsd.edu!swrinde!howland.reston.ans.net!EU.net!ub4b!idefix.CS.kuleuven.ac.be!rc1.vub.ac.be!is3e!philstas From: philstas@vub.ac.be (Stas Philippe) Newsgroups: bionet.molbio.proteins,bionet.xtallography,sci.techniques.xtallography,bionet.immunology,bionet.general Subject: Re: Bionet.molec-model active Followup-To: bionet.molbio.proteins,bionet.xtallography,sci.techniques.xtallography,bionet.immunology,bionet.general Date: 5 Jul 1994 15:08:09 GMT Organization: Brussels Free Universities (VUB/ULB), Belgium Lines: 9 Message-ID: <2vbt0p$kst@rc1.vub.ac.be> References: <2us1tc$h2r@rc1.vub.ac.be> NNTP-Posting-Host: is3e.vub.ac.be X-Newsreader: TIN [version 1.2 PL2] Xref: biosci bionet.molbio.proteins:2181 bionet.xtallography:1004 sci.techniques.xtallography:510 bionet.immunology:1625 bionet.general:10235 Stas Philippe (philstas@vub.ac.be) wrote: overwhelming amount of YES votes. The group is now active and waiting for all molecular modellers to start discussions. Philippe Stas Discussion Leader Philstas@vub.ac.be From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!agate!howland.reston.ans.net!noc.near.net!usenet.elf.com!rpi!rebecca.its.rpi.edu!dressm From: Mark J. Dresser Newsgroups: bionet.molbio.proteins Subject: QUESTIONS: alpha-helix "signals" in proteins Date: 5 Jul 1994 13:29:02 GMT Organization: Swiss Federal Institute of Technology Z"urich Lines: 30 Sender: dressm@rebecca.its.rpi.edu (Mark J. Dresser) Distribution: world Message-ID: <2vbn6u$j4e@usenet.rpi.edu> Reply-To: chs211@stud.chem.ethz.ch NNTP-Posting-Host: rebecca.its.rpi.edu BACKGROUND INFO: In a few weeks i will be giving a presentation at a literature group meeting for the students taking the Biological Chemie Praktikum. (I am one of the students). I have chosen the topic of alpha-helix formation in protiens and will use several papers by G.D. Rose (Rose G.D. et al. (1994) "Rules for alpha-Helix Termination by Glycine" _Science_, 264, 1126; Rose G.D. et al. (1993) "Helix Stop Signals in Proteins and Peptides: The Capping Box" _Biochemistry_, 32, 7605; Rose G.D. et al. "Helix Signals in Proteins" _Science_ 240, 1632) as the main sources of information. QUESTIONS: What role do/could tertiary interactions play in stabilizing the alpha-helix? Does a protein first fold into its secondary structure elements and then into its tertiary structure, afterwhich the original secondary structure elements remain as before the folding into the tertiary structure? (If yes or no, could you please provide references). Rose proposes that "the folded structure of a protein is encrypted in its aa secquence, written in a *code* that remians obscure". (Rose, G.D. et al. (1994) _Science_ 264, 1126). What is the status of this obscure *code* that he is referring to? Is the existence of this *code* a widely held belief? thanks in advance, Mark Dresser e-mail: chs211@stud.chem.ethz.ch From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!banyo!kenp From: kenp@banyo (Kenneth Prehoda) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 5 Jul 1994 20:12:04 GMT Organization: Division of Information Technology Lines: 74 Message-ID: <2vceqk$g8t@news.doit.wisc.edu> References: <2vbn6u$j4e@usenet.rpi.edu> <2vbqh7$6e8@news.doit.wisc.edu> <2vc5a4$gt9@lyra.csx.cam.ac.uk> NNTP-Posting-Host: banyo.nmrfam.wisc.edu X-Newsreader: TIN [version 1.2 PL2] Simon Brocklehurst (Bioc) (smb18@mole.bio.cam.ac.uk) wrote: : Ken Prehoda writes: : >Since protein folding is believed to be thermodynamically controlled : Errr... NOT! What is the problem with my statement. The current consensus is that protein folding is thermodynamically controlled. It is true there are dissentions from the consensus, but when is that not the case? If you believe otherwise, please provide references (and not quotes from Wayne and Garth as you seem to prefer). I refer you to reviews by Baldwin & Kim, Dill, and many others. : >(OK, see Agard, et al. for counterviews), this is really irrelevant : >to what you seem to be getting at. Remember that G is a state function : >and is therefore independent of path. : The bottom line is that folding pathway(s) is/are almost certainly : involved in getting to the _native_ state - so kinetics _are_ : important. : : I would bet money that anyone who says different is wrong! Of course a folding pathway is involved in going from the unfolded state to the native state. That's a given. Whether or not the pathway determines the structure is a very different question. And whether or not you would bet money on it has little to do with the scientific merits of your position. : As for the original point about the relatiave importance of secondary : vs tertiary vs intrinsic interactions, for controlling formation of : (secondary) structure: Could you please explain what you mean by "intrinsic" interactions? If you are refering to the so-called helical-propensity, then how can you distinguish between secondary, tertiary and intrinsic interactions? : Why can't they all be "equally" important??? That is, maybe in : one bit of a protein, intrinsic preferences are crucial for : driving structure formation, whereas in a different bit, secondary : interactions could be dominant. : If this was true, then there would be different percentages : of residues whose conformation was mainly determined by one of : the above "things". What these numbers would be is anyone's guess. : But, it is worth remembering that the so-called "hydrophobic effect" : is most likely to be the major driving force in structure formation. Depending on your definition of the hydrophobic effect, this is highly debatable (well it is debatable regardless of your definition, but without knowing what you mean by the hydrophobic effect I cannot argue the point). : In the simplest model for considering this effect, there need be no : difference between secondary and tertiary interactions when considering : this "effect". : My guess is that, on average, secondary and tertiary interactions of : residues dominate over "intrinsic" residue preferences" in driving the : formation of secondary structure. : _________________________________________________________________________ : | : | ,_ o Simon M. Brocklehurst, : | / //\, Oxford Centre for Molecular Sciences, : | \>> | Department of Biochemistry, University of Oxford, : | \\, Oxford, UK. : | E-mail: smb@bioch.ox.ac.uk : |________________________________________________________________________ -Ken Prehoda kenp@nmrfam.wisc.edu From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!elroy.jpl.nasa.gov!lll-winken.llnl.gov!fastrac.llnl.gov!osi-east2.es.net!pnl-oracle!netnews.nwnet.net!mule.fhcrc.org!fred!schen From: schen@fred (Shu-Chih Chen) Newsgroups: bionet.molbio.proteins Subject: databank for the sizes of proteins Date: 5 Jul 1994 18:34:20 GMT Organization: Fred Hutchinson Cancer Research Center Lines: 16 Message-ID: <2vc93c$uje@mule.fhcrc.org> NNTP-Posting-Host: fred.fhcrc.org X-Newsreader: TIN [version 1.2 PL2] Hi: B A B We observed a change in the abundance for a protein in the low 30kD region, and am interested in knowing what the protien could be. It is in a mammalian cell line. My question is whether there is a databank in which one could search by the sizes of protiens. If not, would anyone be interested in helping constructing one. We could do this by cooperating with the preexisting databank servers. Any information and comments are welcomed. Shu-Chih Chen schen@fred.fhcrc.org p.s. please send info to my E-mail address directly, thanks. From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!howland.reston.ans.net!usenet.ins.cwru.edu!po.CWRU.Edu!jjc4 From: jjc4@po.CWRU.Edu (James J. Campanella) Newsgroups: bionet.molbio.proteins Subject: Needed: Plant actin Date: 5 Jul 1994 17:45:09 GMT Organization: Case Western Reserve University, Cleveland, Ohio (USA) Lines: 17 Message-ID: <2vc675$jrb@usenet.INS.CWRU.Edu> NNTP-Posting-Host: slc8.ins.cwru.edu Hi, My colleagues and I have cloned an actin-like protein from Arabidopsis that has a novel activity. The protein produced by our isolated cDNA is obviously similar to actin but it also has major structural differences-- so we do not believe it to be an Arabidopsis actin. What we would like to do is to test real Arabidopsis actin as a control to demonstrate that it does (or doesn't) have the same novel activity. Unfortunately we do not have any Arabidopsis or plant actin to perform this experiment. Does anyone out in netland have and/or be willing to part with the cDNA for Arabidopsis actin-- or any plant actin in general? Thanx, Jim Campanella -- If you're not part of the solution, you're part of the precipitate. -------------------------------------------------------------------- Jim Campanella, Case Western Reserve University Biology Department Cleveland, Ohio 44106 From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!bjd12 From: bjd12@cus.cam.ac.uk (Ben Davis) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 1994 06:50:36 GMT Organization: University of Cambridge, England Lines: 37 Distribution: world Message-ID: <2vdk7s$ss@lyra.csx.cam.ac.uk> References: <2vbn6u$j4e@usenet.rpi.edu> <2vc5a4$gt9@lyra.csx.cam.ac.uk> <2vceqk$g8t@news.doit.wisc.edu> <2vcoc2$l6i@news.doit.wisc.edu> NNTP-Posting-Host: bootes.cus.cam.ac.uk X-Newsreader: TIN [version 1.2 PL2] Ken Prehoda (kenp@nmrfam.wisc.edu) wrote: : In article <2vbu05$det@lyra.csx.cam.ac.uk> : Ben Davis, bjd12@cus.cam.ac.uk writes: : > I think that more proteins need to be characterised under denaturing : >conditions before the question of structure in unfolded proteins can be : >answered properly. Certainly in the cases where unfolded proteins have : been : >characterised (434 repressor, FK506 BP) structures have been found. They : >tend to be marginal, rather than persistent, but they do seem to be : formed. : >This kind of implies that local interactions can form, and so presumably : >would form early in a folding reaction. : You make a valid point, however, I would suggest that the only : denatured states that we see in the literature are those that have : some type of structure (however little it may be). For proteins that : do have fully unfolded denatured states, there is little incentive : to publish. GIven the amount of work it takes to characterise a denatured state fully (I'm primarily talking full NMR assignment + std biochemical evidence) I'm absolutely sure an example of "totally dentaured protein" would get published, espc. since there aren't any yet ... : -Ken Prehoda : kenp@nmrfam.wisc.edu Ben -- ______________________________________________________________________________ Ben Davis, MRC Protein Function and Design, Cambridge, UK ______________________________________________________________________________ "They can make me do it, but they can't make me do it with dignity." From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!f180-118.net.wisc.edu!kenp From: Ken Prehoda Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 5 Jul 1994 22:54:58 GMT Organization: Univ of Wisc-Madison Lines: 31 Distribution: world Message-ID: <2vcoc2$l6i@news.doit.wisc.edu> References: <2vbn6u$j4e@usenet.rpi.edu> <2vc5a4$gt9@lyra.csx.cam.ac.uk> <2vceqk$g8t@news.doit.wisc.edu> NNTP-Posting-Host: f180-118.net.wisc.edu X-Newsreader: Nuntius Version 1.2 X-XXMessage-ID: X-XXDate: Tue, 5 Jul 1994 23:51:26 GMT In article <2vbu05$det@lyra.csx.cam.ac.uk> Ben Davis, bjd12@cus.cam.ac.uk writes: > I think that more proteins need to be characterised under denaturing >conditions before the question of structure in unfolded proteins can be >answered properly. Certainly in the cases where unfolded proteins have been >characterised (434 repressor, FK506 BP) structures have been found. They >tend to be marginal, rather than persistent, but they do seem to be formed. >This kind of implies that local interactions can form, and so presumably >would form early in a folding reaction. You make a valid point, however, I would suggest that the only denatured states that we see in the literature are those that have some type of structure (however little it may be). For proteins that do have fully unfolded denatured states, there is little incentive to publish. >______________________________________________________________________ _______ > >Ben Davis, >MRC Protein Function and Design, >Cambridge, UK >______________________________________________________________________ _______ > >"They can make me do it, but they can't make me do it with dignity." -Ken Prehoda kenp@nmrfam.wisc.edu From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!NBRF.GEORGETOWN.EDU!POSTMASTER From: POSTMASTER@NBRF.GEORGETOWN.EDU Newsgroups: bionet.molbio.proteins Subject: Re: databank for the sizes of proteins Date: 5 Jul 1994 14:47:44 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HECSWGDMPE95MLUU@NBRF.Georgetown.Edu> NNTP-Posting-Host: net.bio.net In message <2vc93c$uje@mule.fhcrc.org> Shu-Chih Chen (schen@fred.fhcrc.org) asked: > We observed a change in the abundance for a protein in the low 30kD > region, and am interested in knowing what the protien could be. It is > in a mammalian cell line. My question is whether there is a databank > in which one could search by the sizes of protiens. If not, would > anyone be interested in helping constructing one. We could do this > by cooperating with the preexisting databank servers. This information and search capability are already available. The ATLAS program distributed on the CD-ROM with the PIR-International Protein Sequence Data Base provides easily usable capabilities for searching protein sequences by estimated molecular weight ranges in conjunction with other search strategies. The PIR Network Request Server can search for protein sequences by estimated chain length ranges. For more information, request HELP from FILESERV@NBRF.Georgetown.EDU. ------------------------------------------------------------------------ Dr. John S. Garavelli Database Coordinator Protein Information Resource National Biomedical Research Foundation Washington, DC 20007 POSTMAST@GUNBRF.BITNET POSTMASTER@NBRF.GEORGETOWN.EDU From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!smb18 From: smb18@mole.bio.cam.ac.uk (Simon Brocklehurst (Bioc)) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 5 Jul 1994 17:29:40 GMT Organization: University of Cambridge, England Lines: 57 Message-ID: <2vc5a4$gt9@lyra.csx.cam.ac.uk> References: <2vbn6u$j4e@usenet.rpi.edu> <2vbqh7$6e8@news.doit.wisc.edu> NNTP-Posting-Host: mole.bio.cam.ac.uk Ken Prehoda writes: >Since protein folding is believed to be thermodynamically controlled Errr... NOT! >(OK, see Agard, et al. for counterviews), this is really irrelevant >to what you seem to be getting at. Remember that G is a state function >and is therefore independent of path. The bottom line is that folding pathway(s) is/are almost certainly involved in getting to the _native_ state - so kinetics _are_ important. I would bet money that anyone who says different is wrong! As for the original point about the relatiave importance of secondary vs tertiary vs intrinsic interactions, for controlling formation of (secondary) structure: Why can't they all be "equally" important??? That is, maybe in one bit of a protein, intrinsic preferences are crucial for driving structure formation, whereas in a different bit, secondary interactions could be dominant. If this was true, then there would be different percentages of residues whose conformation was mainly determined by one of the above "things". What these numbers would be is anyone's guess. But, it is worth remembering that the so-called "hydrophobic effect" is most likely to be the major driving force in structure formation. In the simplest model for considering this effect, there need be no difference between secondary and tertiary interactions when considering this "effect". My guess is that, on average, secondary and tertiary interactions of residues dominate over "intrinsic" residue preferences" in driving the formation of secondary structure. _________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences, | \>> | Department of Biochemistry, University of Oxford, | \\, Oxford, UK. | E-mail: smb@bioch.ox.ac.uk |________________________________________________________________________ From owner-proteins@net.bio.net Mon Jul 04 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!howland.reston.ans.net!vixen.cso.uiuc.edu!newsrelay.iastate.edu!newsxfer.itd.umich.edu!jobone!lynx.unm.edu!dns1.NMSU.Edu!smori From: smori@nmsu.edu (Shahram Mori) Newsgroups: bionet.molbio.proteins Subject: Discussions on Bionet.Immunology Date: 2 Jul 1994 05:27:06 GMT Organization: New Mexico State University, Las Cruces, NM Lines: 25 Message-ID: <2v2tra$ngp@dns1.NMSU.Edu> NNTP-Posting-Host: dante.nmsu.edu X-Newsreader: TIN [version 1.2 PL1] [ Article crossposted from bionet.molbio.methds-reagnts ] [ Author was Shahram Mori ] [ Posted on 2 Jul 1994 05:09:39 GMT ] Dear netters, I am posting here to let the beloved researchers know that we are trying to start an Immunology discussion group in Bionet.Immunology. Please e-mail Matt Buchanan about the topic that you are interested in. He has posted regarding this in bionet.immunology and his e-mail address can be reached there, or else post your title there. This will be a great opportunity to get interactions on this fascinating field. Hope to see your post there. Cheers, Shahram Mori Program in Molecular Biology Dept of Chemistry and Biochemistry Box 3C NMSU Las Cruces NM 88003 _/\_ _\ /_ \_ _/ || From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!MODEL.PHR.UTEXAS.EDU!rhodes From: rhodes@MODEL.PHR.UTEXAS.EDU ("David G. Rhodes") Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 1994 15:01:13 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 41 Sender: daemon@net.bio.net Distribution: world Message-ID: <9407061701.ZM22872@model.phr.utexas.edu> NNTP-Posting-Host: net.bio.net On Jul 6, 8:49pm, Simon Brocklehurst (Bioc) wrote: > Subject: Re: QUESTIONS: alpha-helix "signals" in proteins > kenp@banyo (Kenneth Prehoda) writes: > > >Simon Brocklehurst (Bioc) (smb18@mole.bio.cam.ac.uk) wrote: > >: Ken Prehoda writes: > ....lots of other stuff deleted ... > > I am arguing that the end state you see depends on the pathway > you are allowed to go down. The important point on the pathway > being, of course, the transition state. So from where I stand, > the idea that hydrophobic collapse happens before hydrogen > bond formation is extremely relevant: the early stages of folding being, > all about pushing the protein towards the transition state. > ______________________________________________________________________ > | > | ,_ o Simon M. Brocklehurst, > | / //\, Oxford Centre for Molecular Sciences, > | \>> | Department of Biochemistry, University of Oxford, > | \\, Oxford, UK. > | E-mail: smb@bioch.ox.ac.uk > |_____________________________________________________________________ > >-- End of excerpt from Simon Brocklehurst (Bioc) Has anyone else noticed the appropriateness of the sig here?? Many of us have used the hiker or skier analogy in discussions of approaches to local or global energy minima. Imho, these arguments work pretty well. -- _____________________________________________________________________ | David G. Rhodes | RHODES@MODEL.PHR.UTEXAS.EDU | | Pharmaceutics Division | | | College of Pharmacy | Phone:(512)471-4681 | | The University of Texas at Austin | Fax: (512)471-7474 }:) | | Austin, TX 78712-1074 | | |___________________________________|_______________________________| From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!agate!lhom From: lhom@OCF.Berkeley.EDU (Louis Hom) Newsgroups: bionet.molbio.proteins Subject: Re: Why only L amino acids? Date: 6 Jul 1994 23:52:15 GMT Organization: U.C. Berkeley Open Computing Facility Lines: 13 Message-ID: <2vfg3f$qca@agate.berkeley.edu> References: <940706182319.1193@jason.uthct.edu> NNTP-Posting-Host: sandstorm.berkeley.edu I think you can invoke an argument of parsimony in this explanation; selection should favor the simplest system. Our present repertoire of enzymes and structural proteins do very well within the limits of 20-ish amino acid forms. Our systems accomplish what they need to accomplish under these circumstances. It would take a lot more components to deal with synthesizing a lot of D aa's as well as L aa's, plus you would have to activate the tRNAs and modify the meanings of codons too. It's a lot of work for what appears to be superfluous variation. -- **************************************************************************** * Lou Hom * "All folks are family." * * lhom@ocf.berkeley.edu * -- John Saponara * **************************************************************************** From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!JASON.UTHCT.EDU!SHAUN From: SHAUN@JASON.UTHCT.EDU ("Shaun D. Black") Newsgroups: bionet.molbio.proteins Subject: Re: Why only L amino acids? Date: 6 Jul 1994 16:18:46 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: <940706182319.1193@jason.uthct.edu> NNTP-Posting-Host: net.bio.net > 6566friedman@vmsa.csd.mu.edu on 6-JUL-1994 15:40 : > > Does anyone have a plausible hypothesis to explain why only L amino > acids are used in proteins? I am teaching an introductory course in > biochemistry this summer and this question was raised by a student. > Please send your answers to: 6566FRIEDMAN@VMS.CSD.MU.EDU > > Alan Friedman Dept Biology Marquette University Milwaukee, WI > Since no experiments can really be done to test the origins of only L-alpha- amino acids in proteins, I would suggest a simple answer, "We don't know." On the other hand, studies on L- and D- amino acids in peptides/proteins exist plentifully. They show, for example, that a helix of L-amino acids folds to yield a right-handed structure, whereas the identical sequence in D-amino acids folds to yield the opposite hand (left handed helix). Finally, you might also point out that D-amino acids do exist 'naturally'. Glycine has no stereochemistry at the alpha carbon, but is found to assume phi/psi angles permitted for both D- and L- amino acids. Also, take a look at the cyclic peptide Gramicidin-S which contains D-Phe. D-amino acids may also form at certain residues in senescent proteins. Enjoy, Shaun (shaun@jason.uthct.edu) From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!bjd12 From: bjd12@cus.cam.ac.uk (Ben Davis) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 1994 16:04:26 GMT Organization: University of Cambridge, England Lines: 67 Distribution: world Message-ID: <2vekma$c3i@lyra.csx.cam.ac.uk> References: <2vbn6u$j4e@usenet.rpi.edu> <2vcoc2$l6i@news.doit.wisc.edu> <2vdk7s$ss@lyra.csx.cam.ac.uk> <2ve3sq$68q@lyra.csx.cam.ac.uk> <2vehcd$hpd@news.doit.wisc.edu> NNTP-Posting-Host: grus.cus.cam.ac.uk X-Newsreader: TIN [version 1.2 PL2] Ken Prehoda (kenp@nmrfam.wisc.edu) wrote: : In article <2vdk7s$ss@lyra.csx.cam.ac.uk> : Ben Davis, bjd12@cus.cam.ac.uk writes: : > GIven the amount of work it takes to characterise a denatured state : >fully (I'm primarily talking full NMR assignment + std biochemical : evidence) : >I'm absolutely sure an example of "totally dentaured protein" would get : >published, espc. since there aren't any yet ... : How can you get a full NMR assignment of a fully denatured protein? : There have been many attempts to study denatured proteins by NMR. : The problem is there is no dispersion (in sharp contrast to the folded : state) : in the signals making it impossible to assign them (indicating : that there is little, if any structure). One would expect that if : there were local interactions in the unfolded state, they would : cause dispersion in the chemical shift. OK. You can get an assignment of proteins under denaturing conditions - infact, its what I've spent the last year or so doing. Two main ways of doing it: (1) Use magnetisation transfer from folded ---> unfolded resonances - ie get a mixture of folded and unfolded protein (1:1), get correlations between the same atom in each state (this is Neri & Wuthrich, 1992, Science 257,1559-1563, also later again in JMB). This is a tricky way to do it - lot of aggregation problems, very crowded spectra, but you can do it. (2) use 13C/15N labelled protein, and triple resonance expts - done recently for FK506 Binding Protein (Logan et al, 1994, JMB, 236, 637-648). Much better way to do it IMHO - use the dispersion in all 3 dimensions. You can also do quite a lot with just 15N labelled protein if the protein is small enough. What people tend to see is that, for most (say > 80%) of the protein, the assignments are close to random coil values (remember 15N shift is more sequence dependant that 1H, so you keep most of this dispersion - very useful), but some resonances deviate from random coil, usually by small amounts (say approx 0.2ppm max for non-labile protons, 0.4ppm for labile). Tie this in with NOE data, you get evidence for non-random coil structures being adopted under "denaturing" conditions. : Maybe it would help if you would explain what would satisfy a "totally : denatured protein?" Very good question - can you ever get a totally random coil protein ? I suspect under say 6M GuHCl you'd be looking at "totally" denatured, since all the aa would be interacting with solvent rather than other aa (hopefully). As it is, proteins denatured by acid, by urea, by heat, by truncation seem to show residual or marginal elements of structure. My earlier point was that it'd be good to get an example of a protein that *was* totally denatured - just as we need more folded structures, so we need more examples of how proteins behace under denaturing conditions. : kenp@nmrfam.wisc.edu -- ______________________________________________________________________________ Ben Davis, MRC Protein Function and Design, Cambridge, UK ______________________________________________________________________________ "They can make me do it, but they can't make me do it with dignity." From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!f181-122.net.wisc.edu!kenp From: Ken Prehoda Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 1994 15:45:00 GMT Organization: Univ of Wisc-Madison Lines: 112 Distribution: world Message-ID: <2vejhs$hpd@news.doit.wisc.edu> References: <2vbn6u$j4e@usenet.rpi.edu> <2vcoc2$l6i@news.doit.wisc.edu> <2vdk7s$ss@lyra.csx.cam.ac.uk> <2ve3sq$68q@lyra.csx.cam.ac.uk> NNTP-Posting-Host: f181-122.net.wisc.edu X-Newsreader: Nuntius Version 1.2 X-XXMessage-ID: X-XXDate: Wed, 6 Jul 1994 16:41:27 GMT In article <2ve3sq$68q@lyra.csx.cam.ac.uk> Simon Brocklehurst, smb18@mole.bio.cam.ac.uk writes: >Excellent, some debate at last on this newsgroup! >(Let's keep it light hearted though!!!) Sounds great, but I would appreciate it if you practiced what you preached. Specifically comments like "Errr....NOT!" can be take a number of ways, some of which may be insulting. That said... >>What is the problem with my statement. The current consensus is >>that protein folding is thermodynamically controlled. > > The problem is that there is simply not a consensus! Well let's look at one example (which is among many): the *widely cited* review by Ken Dill in Biochemistry (Dill, K. (1990) Biochemistry 29, 2357). Dill concludes that the thermodynamic hypothesis can be taken as a given. As I stated in my original reply, there are disagreements over this. > What about the work of Fersht's group, Dobson's group etc. >There seems to be a compulsory order by which bits of structure form on >folding pathways of different proteins. For many proteins, >highly populated kinetic intermediates seems to exist on folding >pathways (although there is some recent debate about this [see >Englander and coworkers]). > > Thus it seems probable (to me at least) that only kinetically >accessible energy minima will be found on the free energy surface. > > Given all this, I don't see why you are so dismissive of the >idea that kinetics are important in protein folding. Even if there is a compulsory order to protein folding, this does not disprove the thermodynamic hypothesis. Neither does folding intermediates. Even so, I stick my neck out so far as to say who cares if protein folding is kinetically controlled? If protein folding is kinetically controlled, it means that if we place a jar of protein on our lab bench, it will eventually find that global free energy minumum. Has this been observed? Not to my knowledge. Now the rate constants may be so small that it will just may take lifetimes or more to observe. Even so, to me this says that for all _practical_ purposes there is thermodynamic control. >>Could you please explain what you mean by "intrinsic" interactions? If >>you are refering to the so-called helical-propensity, then how can >>you distinguish between secondary, tertiary and intrinsic interactions? > > > 1) Intrinsic propensity > > Some people think that particular residues have intrinsic > propensities to exhibit particular conformations. For > example, you will find many references in the literature > saying that alanine residues are strongly helix forming. > > This propensity could be due to the wave function for > alanine. Certainly, it is often difficult to identify [etc...] Like I said before, I cannot see a distinction between "intrinsic propensity" and secondary,tertiary interactions. Let's take as an example alanine which has a high helical propensity. Why does it have a high helical propensity? The most likely explanation is that it does not have a gamma constituent to provide steric constraints. If so, then this cannot be separated from secondary or tertiary considerations. >>Depending on your definition of the hydrophobic effect, this is highly >>debatable. > > Well it doesn't matter how _I_ define it! The point is that, >somehow, it seems likely that intra/inter molecular interactions >involving hydrophobic groups are important in directing folding. > > I agree that there is debate is some quarters about this. But >a lot of the other suggestions (e.g. main-chain hydrogen bond formation >directs folding) are just nonsense. Just like the genetic information of the cell being contained in DNA is nonsense ? What are your arguments against hydrogen bonding "directing" folding? While I personally agree with you that the "hydrophobic-effect" is the dominant contribution to protein stability, there are some very compelling arguments for hydrogen bonding. If you are not aware of these, and are interested, please let me know. As for your definition of the hydrophobic effect, it is very important since you may be discussing the price of tea in China while I am complaining about Apple's stock price. OK, this is obviously getting too long so the hydrophobic effect will have to come in a later post. > _________________________________________________________________________ > | > | ,_ o Simon M. Brocklehurst, > | / //\, Oxford Centre for Molecular Sciences, > | \>> | Department of Biochemistry, University of Oxford, > | \\, Oxford, UK. > | E-mail: smb@bioch.ox.ac.uk > |________________________________________________________________________ -Ken Prehoda kenp@nmrfam.wisc.edu From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!f181-122.net.wisc.edu!kenp From: Ken Prehoda Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 1994 15:07:57 GMT Organization: Univ of Wisc-Madison Lines: 31 Distribution: world Message-ID: <2vehcd$hpd@news.doit.wisc.edu> References: <2vbn6u$j4e@usenet.rpi.edu> <2vcoc2$l6i@news.doit.wisc.edu> <2vdk7s$ss@lyra.csx.cam.ac.uk> <2ve3sq$68q@lyra.csx.cam.ac.uk> NNTP-Posting-Host: f181-122.net.wisc.edu X-Newsreader: Nuntius Version 1.2 X-XXMessage-ID: X-XXDate: Wed, 6 Jul 1994 16:04:25 GMT In article <2vdk7s$ss@lyra.csx.cam.ac.uk> Ben Davis, bjd12@cus.cam.ac.uk writes: > GIven the amount of work it takes to characterise a denatured state >fully (I'm primarily talking full NMR assignment + std biochemical evidence) >I'm absolutely sure an example of "totally dentaured protein" would get >published, espc. since there aren't any yet ... How can you get a full NMR assignment of a fully denatured protein? There have been many attempts to study denatured proteins by NMR. The problem is there is no dispersion (in sharp contrast to the folded state) in the signals making it impossible to assign them (indicating that there is little, if any structure). One would expect that if there were local interactions in the unfolded state, they would cause dispersion in the chemical shift. Maybe it would help if you would explain what would satisfy a "totally denatured protein?" >______________________________________________________________________ _______ > >Ben Davis, >MRC Protein Function and Design, >Cambridge, UK >______________________________________________________________________ _______ -Ken Prehoda kenp@nmrfam.wisc.edu From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!MITVMA.MIT.EDU!W_SHAO%UVMVAX.BITNET From: W_SHAO%UVMVAX.BITNET@MITVMA.MIT.EDU ("Wei Shao\", ADDRESS: IN%\"w_shao@uvmvax.uvm.edu") Newsgroups: bionet.molbio.proteins Subject: signoff Date: 6 Jul 1994 06:39:39 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HEDQTLA0M800188D@uvmvax.uvm.edu> NNTP-Posting-Host: net.bio.net signoff From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!agate!doc.ic.ac.uk!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!smb18 From: smb18@mole.bio.cam.ac.uk (Simon Brocklehurst (Bioc)) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 1994 11:17:46 GMT Organization: University of Cambridge, England Lines: 109 Message-ID: <2ve3sq$68q@lyra.csx.cam.ac.uk> References: <2vbn6u$j4e@usenet.rpi.edu> <2vbqh7$6e8@news.doit.wisc.edu> <2vc5a4$gt9@lyra.csx.cam.ac.uk> <2vceqk$g8t@news.doit.wisc.edu> NNTP-Posting-Host: mole.bio.cam.ac.uk kenp@banyo (Kenneth Prehoda) writes: Excellent, some debate at last on this newsgroup! (Let's keep it light hearted though!!!) So to take some of the points you made: >Simon Brocklehurst (Bioc) (smb18@mole.bio.cam.ac.uk) wrote: >: Ken Prehoda wrote: >: >Since protein folding is believed to be thermodynamically controlled >: Errr... NOT! >What is the problem with my statement. The current consensus is >that protein folding is thermodynamically controlled. The problem is that there is simply not a consensus! > It is true >there are dissentions from the consensus, but when is that not >the case? If you believe otherwise, please provide references (and >not quotes from Wayne and Garth as you seem to prefer). I refer >you to reviews by Baldwin & Kim, Dill, and many others. I'll quote some experiments/refs below. (stuff deleted) >Of course a folding pathway is involved in going from the unfolded >state to the native state. That's a given. Whether or not >the pathway determines the structure is a very different question. What about the work of Fersht's group, Dobson's group etc. There seems to be a compulsory order by which bits of structure form on folding pathways of different proteins. For many proteins, highly populated kinetic intermediates seems to exist on folding pathways (although there is some recent debate about this [see Englander and coworkers]). Thus it seems probable (to me at least) that only kinetically accessible energy minima will be found on the free energy surface. Given all this, I don't see why you are so dismissive of the idea that kinetics are important in protein folding. >: As for the original point about the relatiave importance of secondary >: vs tertiary vs intrinsic interactions, for controlling formation of >: (secondary) structure: >Could you please explain what you mean by "intrinsic" interactions? If >you are refering to the so-called helical-propensity, then how can >you distinguish between secondary, tertiary and intrinsic interactions? 1) Intrinsic propensity Some people think that particular residues have intrinsic propensities to exhibit particular conformations. For example, you will find many references in the literature saying that alanine residues are strongly helix forming. This propensity could be due to the wave function for alanine. Certainly, it is often difficult to identify (from inspection of 3-D structures) inter-residue interactions involving surface alanine residue that could be regarded as strongly stabilizing. 2) Secondary and tertiary interactions The distinction is trivial. Stabilzing inter-residue interactions made within a given secondary structural motif are secondary interactions. Others are tertiary! The question of the point on the folding pathway that interactions become native-like is vigourously debated. (stuff deleted) >: But, it is worth remembering that the so-called "hydrophobic effect" >: is most likely to be the major driving force in structure formation. >Depending on your definition of the hydrophobic effect, this is highly >debatable. Well it doesn't matter how _I_ define it! The point is that, somehow, it seems likely that intra/inter molecular interactions involving hydrophobic groups are important in directing folding. I agree that there is debate is some quarters about this. But a lot of the other suggestions (e.g. main-chain hydrogen bond formation directs folding) are just nonsense. (well it is debatable regardless of your definition, but >without knowing what you mean by the hydrophobic effect I cannot >argue the point). Sure you can! Just say what you think the driving force for finding the native state is (with particular emphasis on things not involving hydrophobic groups). _________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences, | \>> | Department of Biochemistry, University of Oxford, | \\, Oxford, UK. | E-mail: smb@bioch.ox.ac.uk |________________________________________________________________________ From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!pipex!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!smb18 From: smb18@mole.bio.cam.ac.uk (Simon Brocklehurst (Bioc)) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 1994 20:49:17 GMT Organization: University of Cambridge, England Lines: 100 Message-ID: <2vf5cd$i17@lyra.csx.cam.ac.uk> References: <2vbn6u$j4e@usenet.rpi.edu> <2vcoc2$l6i@news.doit.wisc.edu> <2vdk7s$ss@lyra.csx.cam.ac.uk> <2ve3sq$68q@lyra.csx.cam.ac.uk> <2vejhs$hpd@news.doit.wisc.edu> <2veoju$dur@lyra.csx.cam.ac.uk> <2veuvd$o72@news.doit.wisc.edu> NNTP-Posting-Host: mole.bio.cam.ac.uk kenp@banyo (Kenneth Prehoda) writes: >Simon Brocklehurst (Bioc) (smb18@mole.bio.cam.ac.uk) wrote: >: Ken Prehoda writes: >Let's look at what you are saying in a little more detail. I >assume you are saying there is some state which has a free >energy lower than our observed "native" state. However, this is >really irrelevant. Maybe we're talking at cross purposes, 'cos I should have thought that this is extremely relevant to the kinetic/thermodynamic argument. >Between the native and unfolded states >thermodynamic control still holds (i.e. it is path _independent_). >I don't see how you can argue that the folding pathway is >important without having multiple native states. Where are >these other states??? They're kinetically inaccessible. That is, if you don't have transition states of sufficiently low energy to allow the protein to pass through them, then you won't get to a particular energy minimum (native state). I don't see how you can know that there aren't other low energy states on the free energy surface if the protein is being "directed" to fold along a particular pathway. So the question is, how is the protein directed to get to a particular transition state and thence to the native, isn't it? >My impression is that i,i+4 side chains have steric interactions. This >is from discussions with members of Baldwin's group - I can't refer >to any paper's, although they're supposedly out there. You can >easily envision this with a little molecular modelling, though. Well in alpha helices i,i+4 side-chains are close in space, and you do observe a limited number of time-averaged side-chain conformations in such positions (I think Mike Sternberg did some analysis of side-chain dihedral angles a while back - in J. Mol. Biol. ?). But these 'restrictions' do not cause a big problem for helix formation, because there are plenty of conformations available that are favourable. >: (stuff deleted) >: >What are your arguments against hydrogen bonding "directing" folding? >: Here goes: >: 1) There is no thermodynamic (!!) advantage for main-chain polar groups >: to make intramolecular hydrogen bonds rather than to make hydrogen >: bonds with solvent -- is there? >How do you know this? You are assuming amide-amide hydrogen bonds >are equal in strength to amide-water hydrogen bonds. Additionally, >you are neglecting any cooperativy considerations that may be >present. For example, and entropic advantage for hydrogen >bonds is that once one h-bond is formed adjacent hydrogen bonds >no longer must pay that entropic cost. An possible enthalpic >advantage is that once one h-bond is formed, adjacent amides >become more polarized resulting in a more favorable enthalpy. Do you know that whether any/all of these suggestions are correct? >: 2) Unfortunately, for many proteins whose folding pathways have >: been studied so far, it has not been possible to tell if hydrophobic >: collapse occurs before or after the formation of stable, >: long-lived hydrogen bonds. In one case (the protein interleukin >: 1-beta) , though, it was possible to tell: the interpretation is >: difficult, but it seems that hydrophobic collapse occurs _before_ >: the formation of long-lived, stable hydrogen bonds. >So? what does this have to do with the thermodynamic stability of >the protein? The pathway does not determine the stability, but >instead, it is the end states. I hope you aren't arguing against >that? Didn't understand the second to last sentence, so at the risk of repeating myself: I am arguing that the end state you see depends on the pathway you are allowed to go down. The important point on the pathway being, of course, the transition state. So from where I stand, the idea that hydrophobic collapse happens before hydrogen bond formation is extremely relevant: the early stages of folding being, all about pushing the protein towards the transition state. _________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences, | \>> | Department of Biochemistry, University of Oxford, | \\, Oxford, UK. | E-mail: smb@bioch.ox.ac.uk |________________________________________________________________________ From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!agate!spool.mu.edu!vms.csd.mu.edu!6566FRIEDMAN From: 6566friedman@vms.csd.mu.edu Newsgroups: bionet.molbio.proteins Subject: Why only L amino acids? Date: 6 Jul 1994 19:56:35 GMT Organization: Marquette University - Computer Services Lines: 11 Message-ID: <00981067.D5F37D80@vms.csd.mu.edu> Reply-To: 6566friedman@vms.csd.mu.edu NNTP-Posting-Host: vmse.csd.mu.edu Does anyone have a plausible hypothesis to explain why only L amino acids are used in proteins? I am teaching an introductory course in biochemistry this summer and this question was raised by a student. Please send your answers to: 6566FRIEDMAN@VMS.CSD.MU.EDU Alan Friedman Dept Biology Marquette University Milwaukee, WI From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!library.ucla.edu!europa.eng.gtefsd.com!howland.reston.ans.net!torn!thunder!flash!tmcintos From: tmcintos@flash.LakeheadU.Ca (T McIntosh - Biology) Newsgroups: bionet.molbio.proteins Subject: In vitro translation Message-ID: Date: 6 Jul 1994 19:36:18 GMT Sender: news@thunder.LakeheadU.Ca Distribution: bionet Lines: 18 I am in desparate need of help with getting a wheat germ translation system working. I am the 3rd person in this lab to work on it and none of us have been able to get it running. I have used several methods including the Boerhinger Mannheim translation kit and Erickson and Blobel's protocol with and without modifications to precipitation and washing procedures. Boerhinger's protocol calls for discharging tRNas with sodium hydroxide, precipitating with 2% casein?25% TCA and washing unincorporated aas off of GFC filter papers with sodium pyrophosphate in TCA under vacuum filtration. Erickson and Blobel use hot TCA precipitation. I've also used cold TCA precipitation, and several types of filter papers. As far as the actual translation system goes, I've tried changing Wheat germ, mRNA,K, Mg and ATP concentrations to get better incorporation rates. Using either BMV or TMV mRNa I have never gotten more than double the incorporation over the negative control. I have also tried translating a di-ubiquitin mRNa that I transcribed, but with not much better luck.Incorporation goes up at about 20 minutes but by thirty minutes it goes back down and continues to dive. Adding calf thymus tRNA, protease inhibitors and Placental RNase Inhibitors don't help much either. I've even added a wheat germ ribosomal fraction to the system to enrich the concentration of initiation factors. Nothing seems to work. On SDS gels I can see that proteins are being translated above control levels, but it's the kinetics of translation that I'm interested in, so it's incorporation rates that I need. I've used both 14C-leu and 35S-met, and settled on the latter due to overall higher counts. Does anybody have an idiot proof protocol for translation using the wheat germ system that is tried and true? I feel like I must be missing something basic for it not to be working and there is nobody on campus who has any experience with translation systems. If anybody can help please e-mail me. My address is tmmcinto@cs_acad_lan.lakeheadu.ca Tina McIntosh From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!f181-151.net.wisc.edu!kenp From: Ken Prehoda Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 1994 19:30:09 GMT Organization: Univ of Wisc-Madison Lines: 57 Distribution: world Message-ID: <2vf0o1$pbt@news.doit.wisc.edu> References: <2vbn6u$j4e@usenet.rpi.edu> <2ves39$f4f@lyra.csx.cam.ac.uk> <2veuvd$o72@news.doit.wisc.edu> NNTP-Posting-Host: f181-151.net.wisc.edu X-Newsreader: Nuntius Version 1.2 X-XXMessage-ID: X-XXDate: Wed, 6 Jul 1994 20:26:38 GMT In article <2vekma$c3i@lyra.csx.cam.ac.uk> Ben Davis, bjd12@cus.cam.ac.uk writes: > OK. You can get an assignment of proteins under denaturing conditions - >infact, its what I've spent the last year or so doing. Great! My understanding is fairly limited. > Two main ways of doing it: > [techniques deleted] > > What people tend to see is that, for most (say > 80%) of the protein, >the assignments are close to random coil values (remember 15N shift is more >sequence dependant that 1H, so you keep most of this dispersion - very >useful), but some resonances deviate from random coil, usually by small >amounts (say approx 0.2ppm max for non-labile protons, 0.4ppm for labile). >Tie this in with NOE data, you get evidence for non-random coil structures >being adopted under "denaturing" conditions. I have read about this. But to me, this just shows that there is very little structure in the unfolded state. I mean .2ppm max deviation seems as strong support for fully unfolded. As far as NOE's I can't remember any papers describing long range NOE's in an unfolded state. I would appreciate any references. > Very good question - can you ever get a totally random coil protein ? I >suspect under say 6M GuHCl you'd be looking at "totally" denatured, since >all the aa would be interacting with solvent rather than other aa >(hopefully). As it is, proteins denatured by acid, by urea, by heat, by >truncation seem to show residual or marginal elements of structure. It depends greatly on the protein. Many proteins will not unfold in 6M GuHCl. > My earlier point was that it'd be good to get an example of a protein >that *was* totally denatured - just as we need more folded structures, so we >need more examples of how proteins behace under denaturing conditions. Like I said before, I can't imagine a paper where the authors simply describe a denatured state that has no dispersion. Sounds pretty boring but maybe that's just me? >______________________________________________________________________ _______ > >Ben Davis, >MRC Protein Function and Design, >Cambridge, UK >______________________________________________________________________ _______ -Ken Prehoda kenp@nmrfam.wisc.edu From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!banyo!kenp From: kenp@banyo (Kenneth Prehoda) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 1994 18:59:57 GMT Organization: Division of Information Technology Lines: 100 Message-ID: <2veuvd$o72@news.doit.wisc.edu> References: <2vbn6u$j4e@usenet.rpi.edu> <2vcoc2$l6i@news.doit.wisc.edu> <2vdk7s$ss@lyra.csx.cam.ac.uk> <2ve3sq$68q@lyra.csx.cam.ac.uk> <2vejhs$hpd@news.doit.wisc.edu> <2veoju$dur@lyra.csx.cam.ac.uk> NNTP-Posting-Host: banyo.nmrfam.wisc.edu X-Newsreader: TIN [version 1.2 PL2] Simon Brocklehurst (Bioc) (smb18@mole.bio.cam.ac.uk) wrote: : Ken Prehoda writes: : (stuff deleted) : I can see I won't be able to persuade you that kinetics might : be important! Let's look at what you are saying in a little more detail. I assume you are saying there is some state which has a free energy lower than our observed "native" state. However, this is really irrelevant. Between the native and unfolded states thermodynamic control still holds (i.e. it is path _independent_). I don't see how you can argue that the folding pathway is important without having multiple native states. Where are these other states??? : As an aside to your point about keeping proteins in jars on benches. : I'm sure you know that a lot of enzymes loose activity over time. : Some people have postulated that when this happens, the proteins are : going into a deep energy minimum, becoming less flexible, and thus : loosing activity. Good point - one that I carefully neglected to mention ;-) But for now, it is anybody's guess what is happening here. : >Like I said before, I cannot see a distinction between "intrinsic : >propensity" and secondary,tertiary interactions. Let's take as an : >example alanine which has a high helical propensity. Why does it : >have a high helical propensity? The most likely explanation is that : >it does not have a gamma constituent to provide steric constraints. : Why would the absence of a gamma constituent make alanine residues : helix forming, as opposed to beta-strand forming, as opposed to irregular : conformation forming, as opposed to more flexible than larger amino-acids : etc etc? There is plenty of space on the surface of helices to allow : large-side-chains to rotate rapidly in solution. My impression is that i,i+4 side chains have steric interactions. This is from discussions with members of Baldwin's group - I can't refer to any paper's, although they're supposedly out there. You can easily envision this with a little molecular modelling, though. : (stuff deleted) : >What are your arguments against hydrogen bonding "directing" folding? : Here goes: : 1) There is no thermodynamic (!!) advantage for main-chain polar groups : to make intramolecular hydrogen bonds rather than to make hydrogen : bonds with solvent -- is there? How do you know this? You are assuming amide-amide hydrogen bonds are equal in strength to amide-water hydrogen bonds. Additionally, you are neglecting any cooperativy considerations that may be present. For example, and entropic advantage for hydrogen bonds is that once one h-bond is formed adjacent hydrogen bonds no longer must pay that entropic cost. An possible enthalpic advantage is that once one h-bond is formed, adjacent amides become more polarized resulting in a more favorable enthalpy. Both of your assumptions are questionable. : 2) Unfortunately, for many proteins whose folding pathways have : been studied so far, it has not been possible to tell if hydrophobic : collapse occurs before or after the formation of stable, : long-lived hydrogen bonds. In one case (the protein interleukin : 1-beta) , though, it was possible to tell: the interpretation is : difficult, but it seems that hydrophobic collapse occurs _before_ : the formation of long-lived, stable hydrogen bonds. So? what does this have to do with the thermodynamic stability of the protein? The pathway does not determine the stability, but instead, it is the end states. I hope you aren't arguing against that? : 3) Every amino-acid residue (except proline) has the same main-chain : capacity to form main-chain hydrogen bonds - it's the side-chains : that provide the variation. i.e. the sequence of the protein : directs it to fold. : 4) Hydrogen bonds involving surface side-chains seem to be important : at the ends of secondary structural motifs - so-called caps. : But these are not that highly conserved across homologous : families: thus they probably don't drive the folding of the : protein. : _________________________________________________________________________ : | : | ,_ o Simon M. Brocklehurst, : | / //\, Oxford Centre for Molecular Sciences, : | \>> | Department of Biochemistry, University of Oxford, : | \\, Oxford, UK. : | E-mail: smb@bioch.ox.ac.uk : |________________________________________________________________________ -Ken Prehoda kenp@nrmfam.wisc.edu From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!pipex!doc.ic.ac.uk!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!smb18 From: smb18@mole.bio.cam.ac.uk (Simon Brocklehurst (Bioc)) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 1994 18:10:49 GMT Organization: University of Cambridge, England Lines: 27 Message-ID: <2ves39$f4f@lyra.csx.cam.ac.uk> References: <2vbn6u$j4e@usenet.rpi.edu> <2vcoc2$l6i@news.doit.wisc.edu> <2vdk7s$ss@lyra.csx.cam.ac.uk> <2ve3sq$68q@lyra.csx.cam.ac.uk> <2vejhs$hpd@news.doit.wisc.edu> <2veoju$dur@lyra.csx.cam.ac.uk> NNTP-Posting-Host: mole.bio.cam.ac.uk sre@al.cam.ac.uk (Sean Eddy) writes: >In article <2veoju$dur@lyra.csx.cam.ac.uk> smb18@mole.bio.cam.ac.uk (Simon Brocklehurst (Bioc)) writes: > >1) There is no thermodynamic (!!) advantage for main-chain polar groups > > to make intramolecular hydrogen bonds rather than to make hydrogen > > bonds with solvent -- is there? >I've always thought that there was a favorable entropic term. If you >make intramolecular H-bonds, you free up a lot of individual water >molecules that would otherwise be constrained. Even though the newly >freed waters go and H-bond to other waters in solution (so there's no >significant enthalpic contribution) it seems like the number of >degrees of freedom in the system increase, so the entropy increases. >Is this wrong? But the protein chain becomes more constrained when it forms intramolecular hydrogen bonds, compared to when it is making h-bonds with solvent. -- Simon >-- >- Sean Eddy >- MRC Laboratory of Molecular Biology, Cambridge, England >- sre@mrc-lmb.cam.ac.uk From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!usc!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!nntp-serv.cam.ac.uk!sre From: sre@al.cam.ac.uk (Sean Eddy) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 94 18:35:23 Organization: Laboratory of Molecular Biology, MRC, Cambridge UK Lines: 17 Message-ID: References: <2vbn6u$j4e@usenet.rpi.edu> <2vcoc2$l6i@news.doit.wisc.edu> <2vdk7s$ss@lyra.csx.cam.ac.uk> <2ve3sq$68q@lyra.csx.cam.ac.uk> <2vejhs$hpd@news.doit.wisc.edu> <2veoju$dur@lyra.csx.cam.ac.uk> NNTP-Posting-Host: al.mrc-lmb.cam.ac.uk In-reply-to: smb18@mole.bio.cam.ac.uk's message of 6 Jul 1994 17:11:26 GMT In article <2veoju$dur@lyra.csx.cam.ac.uk> smb18@mole.bio.cam.ac.uk (Simon Brocklehurst (Bioc)) writes: >1) There is no thermodynamic (!!) advantage for main-chain polar groups > to make intramolecular hydrogen bonds rather than to make hydrogen > bonds with solvent -- is there? I've always thought that there was a favorable entropic term. If you make intramolecular H-bonds, you free up a lot of individual water molecules that would otherwise be constrained. Even though the newly freed waters go and H-bond to other waters in solution (so there's no significant enthalpic contribution) it seems like the number of degrees of freedom in the system increase, so the entropy increases. Is this wrong? -- - Sean Eddy - MRC Laboratory of Molecular Biology, Cambridge, England - sre@mrc-lmb.cam.ac.uk From owner-proteins@net.bio.net Tue Jul 05 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!library.ucla.edu!europa.eng.gtefsd.com!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!smb18 From: smb18@mole.bio.cam.ac.uk (Simon Brocklehurst (Bioc)) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 6 Jul 1994 17:11:26 GMT Organization: University of Cambridge, England Lines: 64 Message-ID: <2veoju$dur@lyra.csx.cam.ac.uk> References: <2vbn6u$j4e@usenet.rpi.edu> <2vcoc2$l6i@news.doit.wisc.edu> <2vdk7s$ss@lyra.csx.cam.ac.uk> <2ve3sq$68q@lyra.csx.cam.ac.uk> <2vejhs$hpd@news.doit.wisc.edu> NNTP-Posting-Host: mole.bio.cam.ac.uk Ken Prehoda writes: (stuff deleted) I can see I won't be able to persuade you that kinetics might be important! As an aside to your point about keeping proteins in jars on benches. I'm sure you know that a lot of enzymes loose activity over time. Some people have postulated that when this happens, the proteins are going into a deep energy minimum, becoming less flexible, and thus loosing activity. >Like I said before, I cannot see a distinction between "intrinsic >propensity" and secondary,tertiary interactions. Let's take as an >example alanine which has a high helical propensity. Why does it >have a high helical propensity? The most likely explanation is that >it does not have a gamma constituent to provide steric constraints. Why would the absence of a gamma constituent make alanine residues helix forming, as opposed to beta-strand forming, as opposed to irregular conformation forming, as opposed to more flexible than larger amino-acids etc etc? There is plenty of space on the surface of helices to allow large-side-chains to rotate rapidly in solution. (stuff deleted) >What are your arguments against hydrogen bonding "directing" folding? Here goes: 1) There is no thermodynamic (!!) advantage for main-chain polar groups to make intramolecular hydrogen bonds rather than to make hydrogen bonds with solvent -- is there? 2) Unfortunately, for many proteins whose folding pathways have been studied so far, it has not been possible to tell if hydrophobic collapse occurs before or after the formation of stable, long-lived hydrogen bonds. In one case (the protein interleukin 1-beta) , though, it was possible to tell: the interpretation is difficult, but it seems that hydrophobic collapse occurs _before_ the formation of long-lived, stable hydrogen bonds. 3) Every amino-acid residue (except proline) has the same main-chain capacity to form main-chain hydrogen bonds - it's the side-chains that provide the variation. i.e. the sequence of the protein directs it to fold. 4) Hydrogen bonds involving surface side-chains seem to be important at the ends of secondary structural motifs - so-called caps. But these are not that highly conserved across homologous families: thus they probably don't drive the folding of the protein. _________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences, | \>> | Department of Biochemistry, University of Oxford, | \\, Oxford, UK. | E-mail: smb@bioch.ox.ac.uk |________________________________________________________________________ From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!sdd.hp.com!nigel.msen.com!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!ipp-garching.mpg.de!alf.biochem.mpg.de!krasel From: krasel@alf.biochem.mpg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins,bionet.cellbiol,bionet.molbio.methds-reagnts Subject: Antibodies against adenylyl cyclase? Date: 7 Jul 1994 09:09:44 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 14 Message-ID: <2vggooINN2pic@sat.ipp-garching.mpg.de> NNTP-Posting-Host: alf.biochem.mpg.de X-Newsreader: TIN [version 1.1 PL8] Xref: biosci bionet.molbio.proteins:2207 bionet.cellbiol:679 bionet.molbio.methds-reagnts:15988 Hello fellow netters, does anybody of you know about an antibody against adenylyl cyclase which is not subtype specific and suitable for western blotting? Commercial availability would be preferred. Thanks in advance, --Cornelius. -- /* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */ /* email: krasel@alf.biochem.mpg.de fax: +49 89 8578 3795 */ /* "People are DNA's way of making more DNA." (Edward O. Wilson, 1975) */ From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!usc!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!f181-168.net.wisc.edu!kenp From: Ken Prehoda Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 8 Jul 1994 00:23:51 GMT Organization: Univ of Wisc-Madison Lines: 34 Distribution: world Message-ID: <2vi6an$lcm@news.doit.wisc.edu> References: <2vbn6u$j4e@usenet.rpi.edu> <2vhgo5$flp@usenet.rpi.edu> <2vhv54$i94@news.doit.wisc.edu> <2vhqqv$g9f@lyra.csx.cam.ac.uk> NNTP-Posting-Host: f181-168.net.wisc.edu X-Newsreader: Nuntius Version 1.2 X-XXMessage-ID: X-XXDate: Thu, 7 Jul 1994 01:20:20 GMT In article <2vgk5j$2hv@lyra.csx.cam.ac.uk> Ben Davis, bjd12@cus.cam.ac.uk writes: > .2ppm deviation from random coil for a non-labile proton is significant; >any structure present is going to be in fast exchange with other (unfolded) >conformations, so you can't expect massive deviations. The same idea applies >for NOE's - to see a long range NOE, the two protons would have to be in >close proximity for a reasonable length of time, which is unlikely in a >structure which is (at best) marginal. However, this doesn't mean the >protein is unfolded - its not in a random walk conformation - but you do >have to look hard for evidence of structure. These structures also tend to >be loose associations of hydrophobic sidechains, which (if there's no >aromatic residue around) tend to produce only small changes in chemical >shift anyway. Isn't it true that any unfolded structures that have stabilizing interactions will be favored over those that don't? If so, then one would expect that the protein would spend much more _time_ in the stabilized conformation meaning deviations greater than .2ppm. The some goes for NOE's. >______________________________________________________________________ _______ > >Ben Davis, >MRC Protein Function and Design, >Cambridge, UK >______________________________________________________________________ _______ -Ken Prehoda kenp@nmrfam.wisc.edu From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!smb18 From: smb18@mole.bio.cam.ac.uk (Simon Brocklehurst (Bioc)) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 7 Jul 1994 22:39:29 GMT Organization: University of Cambridge, England Lines: 121 Message-ID: <2vi071$hv5@lyra.csx.cam.ac.uk> References: <2vbn6u$j4e@usenet.rpi.edu> <2vcoc2$l6i@news.doit.wisc.edu> <2vdk7s$ss@lyra.csx.cam.ac.uk> <2ve3sq$68q@lyra.csx.cam.ac.uk> <2vejhs$hpd@news.doit.wisc.edu> <2veoju$dur@lyra.csx.cam.ac.uk> <2veuvd$o72@news.doit.wisc.edu> <2vf5cd$i17@lyra.csx.cam.ac.uk> <2vhh69$at6@news.doit.wisc.edu> NNTP-Posting-Host: mole.bio.cam.ac.uk kenp@banyo (Kenneth Prehoda) writes: >: They're kinetically inaccessible. That is, if you don't have >: transition states of sufficiently low energy to allow the protein to >: pass through them, then you won't get to a particular energy minimum >: (native state). >That's my point - they're kinetically inaccessible (in most cases) if >they are there at all. So of what importance are they? Sorry, we're agreed on this point! But I think the pathway from the unfolded to the native state involves going over energy maxima, and I think this must be where our difference opinion is (right or not?). Maybe now's the time to clarify a few points? 1) Do you believe that when proteins fold they, do so along particular pathways that are directed by the sequence? 2) Do you think that these pathways involve traversing energy maxima (transition states)? 3) If yes to the transition state idea, do you think that these states have some native like structure (e.g. partially native secondary structure)? 4) Do you think that rather than going through transition states, there is a roughly downhill path from the unfolded state to the native state i.e. no significant maxima? My answers are: 1) Yes 2) Yes 3) Yes 4) No It seems to me that the yes answer to question 3) is particulary important to arguments about kinetic control? (stuff deleted) (talking about side-chain conformations in helices...) >Well, the researchers in the field that I have talked to believe >that these restrictions do cause energetic differences which is >one contribution the the "propensity." I don't disagree that this is important. It's just that you seemed to imply that alanine residues are helix forming 'cos they don't make any unfavourable steric nteractions. I was just pointing out that some people think otherwise i.e. they think that alanines intrinsically (i.e. on their own!) like to be in a helical conformation. (Personally, I don't have strong opinions on this matter). (stuff deleted) >I wish. See Gellman et al. for extensive discussions. My point >is that the role of hydrogen bonds in protein stability is >entirely _uncertain_ in contrast to your strong opinion otherwise. I never meant to imply that hydrogen bonds were not important contributors to the stability of the native state. I do believe that the hydrophobic side-chains of residues get together at least on the same time-scale as H-bonds form, and quite likely before. Thus I don't see how H-bond formation directs folding. >For one thing, how do you measure the strength of a hydrogen bond? Tricky do this well: You can do protein engineering experiments to _try_ to estimate the strength of hydrogen bonds involving side-chains (see some of Fersht's work). You can monitor rates of hydron exhange of amide protons by solution homo and heteronuclear magnetic resonance spectroscopy. But the interpretation is complicated by motions other than transient breaking of hydrogen bonds. This does give a handle on main-chain H-bonds though. I'm sure there are loads of ways of doing this, but it's late and I can't think of any off hand! Anyway, depending on the conditions the protein is under, the relative strengths of all kinds of non-covalent interactions will change. I don't want to flog a dead horse, but I think the important question about protein folding is: How does the sequence of the protein limit the number of conformations that need to be explored to find the native state? My feeling is that this search involves going uphill as well as downhill on the free energy surface, traversing highly populated intermediates and less highly populated transitions states. Characterising the structures of partly folded states (and even completely "unfolded" [whatever that means] states) is obviously an important step forward to understanding protein folding. Do you really not think that understanding the mechanism(s) by which proteins fold is interesting/important? _________________________________________________________________________ | | ,_ o Simon M. Brocklehurst, | / //\, Oxford Centre for Molecular Sciences, | \>> | Department of Biochemistry, University of Oxford, | \\, Oxford, UK. | E-mail: smb@bioch.ox.ac.uk |________________________________________________________________________ From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!banyo!kenp From: kenp@banyo (Kenneth Prehoda) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 7 Jul 1994 22:21:24 GMT Organization: Division of Information Technology Lines: 42 Message-ID: <2vhv54$i94@news.doit.wisc.edu> References: <2vbn6u$j4e@usenet.rpi.edu> <1994Jul7.162642.35028@ucl.ac.uk> NNTP-Posting-Host: banyo.nmrfam.wisc.edu X-Newsreader: TIN [version 1.2 PL2] Mr Neville Steven Percy (spbcnsp@ucl.ac.uk) wrote: : Loadsa people write: : [...] Loadsa stuff -- an excellent thread! -- [...] [stuff deleted] : >Does a protein first fold into its secondary structure elements and then into its tertiary structure, afterwhich the original secondary structure elements : >remain as before the folding into the tertiary structure? (If yes or no, could : >you please provide references). : Yes, this is correct, but it should further be pointed out that in the lumen of : the rough endoplasmic reticulum where all this folding of the nascent protein : chain is going on, there are a great quantity of chaperone molecules. : Chaperones serve to protect regions of secondary structure from the : potentially damaging effect of exposure to the aqueous environment, until the : entire protein has been synthesized and all the secondary structure elements : can align to one another and form the final tertiary configuration, at which : point the chaperone dis-associates, and leaves this to happen. : I imagine the kinetics of interaction with chaperones is therefore very : important in vivo, if not in such in vitro systems as may be used for protein : structure studies... but this is very IMHO, as there are obviously a lot more : specialized people than myself contributing to this thread! :) It is far from certain that secondary structure forms before the global fold is formed (framework model). The hydrophobic collapse model where nonpolar surface is buried before secondary structure forms is just as likely at this point. Also, as far as I know chaperones are simply catalysts and do not affect the end state of protein folding. The final native state will be the same whether or not a chaperone is present - albeit it much more slowly without. : (Mad Cow Disease) Neville Percy ; spbcnsp@ucl.ac.uk _.=~-.--=~ : -------------------------------------------------------------------------------- : BioMOO, the virtual reality for biologists; telnet bioinfo.weizmann.ac.il 8888 : -------------------------------------------------------------------------------- -Ken Prehoda kenp@nmrfam.wisc.edu From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!agate!library.ucla.edu!europa.eng.gtefsd.com!howland.reston.ans.net!EU.net!Germany.EU.net!netmbx.de!zrz.TU-Berlin.DE!math.fu-berlin.de!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!ipp-garching.mpg.de!nmrvex.biochem.mpg.de!engh From: engh@nmrvex.biochem.mpg.de (Richard Engh) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 7 Jul 1994 08:07:46 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 24 Distribution: world Message-ID: <2vgd4iINN23mt@sat.ipp-garching.mpg.de> References: <9407061701.ZM22872@model.phr.utexas.edu> NNTP-Posting-Host: nmrvex.biochem.mpg.de X-Newsreader: TIN [version 1.1 PL8] This very interesting fundamental discussion is a little bit disguised by the rather more specific subject title... Regarding thermodynamic versus kinetic control of protein folding, I would like to mention the case of some serpin protein inhibitors, in particular plasminogen activator inhibitor, studied by E. Goldsmith and co-workers. PAI has an inhibitory fold in vitro only for a limited time, and then undergoes a transition to a latent state (see Mottonen et al., Nature 355, 270-273, 1992; see also a recent issue of Structure for related serpin work and discussion). In vivo, the active form may be stabilized by complexation. The point is, the primary functional conformation is NOT the global minimum for the isolated protein. Is this kinetic control? Even if the complexed form in vivo IS a global minimum (and I don't know what the current opinion on this is), is it a case where a metastable fold has to be 'captured' and complexed. Any serpin researchers reading this who could comment? -R.Engh From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!MODEL.PHR.UTEXAS.EDU!rhodes From: rhodes@MODEL.PHR.UTEXAS.EDU ("David G. Rhodes") Newsgroups: bionet.molbio.proteins Subject: Re: Why only L amino acids? Date: 7 Jul 1994 11:44:26 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: <9407071344.ZM24336@model.phr.utexas.edu> NNTP-Posting-Host: net.bio.net What about coriolis force as an admittedly weak but chiral force??? }:) Seriously, though... I seem to recall (~4 years ago) a crystallization/precipitation that resulted in a helical structure (Nature paper?). The authors repeated the experiment in a colleague's lab south of the equator, and got the opposite chirality. They could not explain the mechanistic details, as I remember, but suggested that coriolis force might be involved. ...any ideas??? -- _____________________________________________________________________ | David G. Rhodes | RHODES@MODEL.PHR.UTEXAS.EDU | | Pharmaceutics Division | | | College of Pharmacy | Phone:(512)471-4681 | | The University of Texas at Austin | Fax: (512)471-7474 }:) | | Austin, TX 78712-1074 | | |___________________________________|_______________________________| From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!usc!elroy.jpl.nasa.gov!lll-winken.llnl.gov!noc.near.net!usenet.elf.com!rpi!rebecca.its.rpi.edu!dressm From: (Mark J. Dresser) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 7 Jul 1994 18:15:33 GMT Organization: The Swiss Federal Institute of Technology Z"urich Lines: 42 Sender: dressm@rebecca.its.rpi.edu (Mark J. Dresser) Distribution: world Message-ID: <2vhgo5$flp@usenet.rpi.edu> References: <2vbn6u$j4e@usenet.rpi.edu> <1994Jul7.162642.35028@ucl.ac.uk> Reply-To: chs211@stud.chem.ethz.ch NNTP-Posting-Host: rebecca.its.rpi.edu In article <1994Jul7.162642.35028@ucl.ac.uk>, spbcnsp@ucl.ac.uk (Mr Neville Steven Percy) writes: |> Loadsa people write: |> |> [...] Loadsa stuff -- an excellent thread! -- [...] |> |> BUT: Mark J. Dresser wrote: |> |> >QUESTIONS: What role do/could tertiary interactions play in stabilizing |> >the alpha-helix? |> |> Tertiaries are bound to play a strong role, and in fact quaternaries have |> been completely overlooked in this thread so far! It seemed obvious to me that tertiary interactions also could play a very important role in the stabilization of secondary structural elements, which is why i found the papers by G.D. Rose et al somewhat disturbing (see first message in this thread for the references). In particular, in the _Science_ (1988), 240, 1632 ff. paper, statements like "It is possible that helices lacking NTB's and CTP's can nevertheless be stabilized by tertiary interactions, ...". Is it not also possible that tert. interactions stabilize helices that *have* NTP's and CTP's? This question is not adressed in this paper or the more recent articles. Yesterday Simon Brocklehurst wrote that "the distinction [between sec. and tert. interactions] is trivial". I disagree. If tert. interactions do indeed play a strong role in the stablization of helix structures, for example, this should not be ignored, and in my opinion, at least in the papers that i originaly mentioned, it has been. Does anyone know of any research that has been done in this area? If so please let me know. thanks, Mark ------------ Mark Dresser Department of Chemistry The Swiss Federal Institute of Technology Z"urich e-mail: chs211@stud.chem.ethz.ch From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!agate!spool.mu.edu!caen!malgudi.oar.net!news.pipeline.com!jmack.pipeline.com!user From: jmack@pipeline.com (John Mack) Newsgroups: bionet.molbio.proteins Subject: Re: Why only L amino acids? Followup-To: bionet.molbio.proteins Date: Thu, 07 Jul 1994 23:45:45 -0401 Organization: Mack Computer Services Lines: 37 Distribution: world Message-ID: References: <9407071344.ZM24336@model.phr.utexas.edu> NNTP-Posting-Host: jmack.pipeline.com In article <9407071344.ZM24336@model.phr.utexas.edu>, rhodes@MODEL.PHR.UTEXAS.EDU ("David G. Rhodes") wrote: > What about coriolis force as an admittedly weak but chiral force??? > > }:) > > Seriously, though... > I seem to recall (~4 years ago) a crystallization/precipitation that > resulted in a helical structure (Nature paper?). The authors repeated the > experiment in a colleague's lab south of the equator, and got the opposite > chirality. They could not explain the mechanistic details, as I remember, > but suggested that coriolis force might be involved. > > ...any ideas??? Yes. We know L-amino acids fold into right-handed helices. (And vice versa.) Therefore, look for forces (coriolis? asymmetric surface catalysts?) that favor right-handed helices. If right-handed helices are favored in one hemisphere due to the coriolis force, then that may be the hemisphere where these were first incorporated into successful, Darwinian replicating, nano-machines which eventually became living things. Thus, it is just fortuitous that life began in the hemisphere that favored right-handed helices and also L-amino acids. After all, life would be just the same with D-amino acids and left-handed helices. Maybe the L and D nanomachines existed simultaneously on Earth and dominated their respective hemispheres! But, for some unknown reason (a comet striking Earth?) the D nanomachines never developed into living things. The L machines did. The living beings invaded the entire Earth afterward and chaged conditions such that D nanomachines could not compete and never became truly alive! From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!usc!elroy.jpl.nasa.gov!lll-winken.llnl.gov!fnnews.fnal.gov!nntp-server.caltech.edu!rickert From: rickert@cco.caltech.edu (Keith Warren Rickert) Newsgroups: bionet.molbio.proteins Subject: Re: Why only L amino acids? Date: 8 Jul 1994 02:46:59 GMT Organization: California Institute of Technology, Pasadena Lines: 24 Message-ID: <2vien3$bpr@gap.cco.caltech.edu> References: <940706182319.1193@jason.uthct.edu> <2vicci$fdv@nyx10.cs.du.edu> NNTP-Posting-Host: accord.cco.caltech.edu X-Newsreader: NN version 6.5.0 #12 (NOV) In <2vicci$fdv@nyx10.cs.du.edu> pfinerty@nyx10.cs.du.edu (being) writes: >-----BEGIN PGP SIGNED MESSAGE----- >a recent issue of science or nature (i can't remember which) included an >article on experiments using magnetic fields to influence the chirality of >the products of a reaction. the influence is very strong too, something >like 80-90% of the product is of one type. it was suggested that, although >much weaker, the earth's magnetic field might have exerted a similar >effect on the early events of chiral molecule formation. i think they >might also have suggested that a tempory increase in the magnetic field >could also have happened. Unfortunately, those experiments, originally reported in Angewandte Chemie, were shown to have been fabricated, and the retraction will be published shortly. Keith -- Keith Rickert | "Aye, though we hunted high and low, and hunted keith@imppig.caltech.edu | everywhere, of the three men's fate, we found no rickert@cco.caltech.edu | trace, in any time, in any place, but a door ajar, | and an untouched meal, and an overtoppled chair." From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!munnari.oz.au!news.uwa.edu.au!camn From: camn@uniwa.uwa.edu.au (Cameron Neylon) Newsgroups: bionet.molbio.proteins Subject: Re: Why only L amino acids? Date: 8 Jul 1994 01:46:32 GMT Organization: The University of Western Australia Lines: 33 Message-ID: <2vib5o$eue@styx.uwa.edu.au> References: <00981067.D5F37D80@vms.csd.mu.edu> <2vgjmsINN10e8@bio5.chemie.uni-freiburg.de> NNTP-Posting-Host: uniwa.uwa.edu.au X-Newsreader: NN version 6.4.19 #1 dreyer@bio5.chemie.uni-freiburg.de (Matthias Dreyer) writes: >|> Does anyone have a plausible hypothesis to explain why only L amino >|> acids are used in proteins? I am teaching an introductory course in >|> biochemistry this summer and this question was raised by a student. >|> Please send your answers to: >|> > >Schulz & Schirmer (Prinicples of Protein Structure,1985) dropped a few >lines about this problem: >"... Although it had been shown that the intrinsic asymmetric beta-decay >expresses itself as molecular asymmetry by preferentially destroying >D-amino acids, the observed effect of a few percent is too small to explain >the selection. Dawkins in 'The extended Phenotype' refers to a model that shows that a selction pressure of 1% is enough to wipe out a gene in 1000 generations. I don't have the book here but I"m pretty sure he gives a reference. For my two cents worth. It appears from a brief glance at my old textbook (Biochemistry, Stryer, 3rd edition) that the assymetry is induced in all the amino acids at teh point of transamination of the alpha keto acid to the alpha amino acid. The transmainase enzymes are all (presumably) stereospecific, so at least there is a common point,in the 'proto-transmainase' where the selection pressure could have acted to affect all aa. Could be by chance I suppose, but I'd tend to favour something that gave a good reason why one was chosen over the other. From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!tuli!kenp From: kenp@tuli (Kenneth Prehoda) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 8 Jul 1994 01:31:13 GMT Organization: Division of Information Technology Lines: 121 Message-ID: <2via91$ms8@news.doit.wisc.edu> References: <2vbn6u$j4e@usenet.rpi.edu> <2vcoc2$l6i@news.doit.wisc.edu> <2vdk7s$ss@lyra.csx.cam.ac.uk> <2ve3sq$68q@lyra.csx.cam.ac.uk> <2vejhs$hpd@news.doit.wisc.edu> <2veoju$dur@lyra.csx.cam.ac.uk> <2veuvd$o72@news.doit.wisc.edu> <2vf5cd$i17@lyra.csx.cam.ac.uk> <2vhh69$at6@news.doit.wisc.edu> <2vi071$hv5@lyra.csx.cam.ac.uk> NNTP-Posting-Host: tuli.nmrfam.wisc.edu X-Newsreader: TIN [version 1.2 PL2] [sorry about that mistaken post, my newsreader (read: me) made a mistake-kep] Simon Brocklehurst (Bioc) (smb18@mole.bio.cam.ac.uk) wrote: : kenp@banyo (Kenneth Prehoda) writes: : >: They're kinetically inaccessible. That is, if you don't have : >: transition states of sufficiently low energy to allow the protein to : >: pass through them, then you won't get to a particular energy minimum : >: (native state). : >That's my point - they're kinetically inaccessible (in most cases) if : >they are there at all. So of what importance are they? : Sorry, we're agreed on this point! : But I think the pathway from the unfolded to the native state involves : going over energy maxima, and I think this must be where our difference : opinion is (right or not?). You deleted my question: again I ask you, do you believe it is valid to use equilibrium constants when studying protein systems??? If so, I don't see how you can argue kinetic control. Nor did you answer my question concerning the importance of these kinetically inaccessible states (if such a thing exists). As to your second point, _by definition_ the folding pathway involves going over energy maxima. : 1) Do you believe that when proteins fold they, do so along : particular pathways that are directed by the sequence? : 2) Do you think that these pathways involve traversing energy : maxima (transition states)? : 3) If yes to the transition state idea, do you think that these : states have some native like structure (e.g. partially native : secondary structure)? : 4) Do you think that rather than going through transition states, : there is a roughly downhill path from the unfolded state to the : native state i.e. no significant maxima? : My answers are: : 1) Yes : 2) Yes : : 3) Yes : 4) No As I have said before, the overwhelming evidence is that protein folding for most proteins is under thermodynamic control. What this means is that the final structure is COMPLETELY INDEPENDENT OF THE PATHWAY. Of course there is some "transition state" involved in the folding process. However, this determines the _rate_ of folding, not the final structure. As such, the structure of the transition state only affects the rate of folding. There simply isn't a good argument for kinetic control. What we are concerned with is the pathway *between our observed folded state and observed unfolded state*. It is undeniable that equilibrium (for all practical purposes), and therefore thermodynamic control is going on here. Otherwise, we wouldn't be using Keq's to describe the system. : It seems to me that the yes answer to question 3) is particulary : important to arguments about kinetic control? Maybe you could explain. Even if kinetic control is indeed true, the structure of the transition state still determines the rate and not the end state. : >I wish. See Gellman et al. for extensive discussions. My point : >is that the role of hydrogen bonds in protein stability is : >entirely _uncertain_ in contrast to your strong opinion otherwise. : I never meant to imply that hydrogen bonds were not important : contributors to the stability of the native state. That doesn't fit with your previous quote shown below: : I agree that there is debate is some quarters about this. But : a lot of the other suggestions (e.g. main-chain hydrogen bond formation : directs folding) are just nonsense. [back to current post] : I don't want to flog a dead horse, but I think the important question : about protein folding is: : How does the sequence of the protein limit the number of : conformations that need to be explored to find the native state? : My feeling is that this search involves going uphill as well : as downhill on the free energy surface, traversing highly populated : intermediates and less highly populated transitions states. : Characterising the structures of partly folded states (and even : completely "unfolded" [whatever that means] states) is obviously an : important step forward to understanding protein folding. : Do you really not think that understanding the mechanism(s) by : which proteins fold is interesting/important? I believe it is an interesting academic question, but as far as understanding the final protein structure, I believe it is irrelevant. If you have a compelling argument otherwise, I will gladly change that belief. : _________________________________________________________________________ : | : | ,_ o Simon M. Brocklehurst, : | / //\, Oxford Centre for Molecular Sciences, : | \>> | Department of Biochemistry, University of Oxford, : | \\, Oxford, UK. : | E-mail: smb@bioch.ox.ac.uk : |________________________________________________________________________ -Ken Prehoda kenp@nmrfam.wisc.edu From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!usc!nic-nac.CSU.net!newshub.sdsu.edu!sunstroke!pthomas From: pthomas@sunstroke.sdsu.edu (Paul Thomas) Newsgroups: bionet.molbio.proteins Subject: help with 2d gels Date: 8 Jul 1994 01:11:57 GMT Organization: San Diego State University, College of Sciences Lines: 18 Message-ID: <2vi94t$g8r@pandora.sdsu.edu> NNTP-Posting-Host: sunstroke.sdsu.edu Summary: seeking people in San Diego w 2dgel experience for consultation Keywords: 2d gels/proteins/consultation X-Newsreader: TIN [version 1.2 PL2] Hello, I am trying to help a friend locate people with experience in 2d gel analysis of proteins in the San Diego area who could help with advice and consultation. If you might, please send email to me, pthomas@lifsci.sdsu.edu or to, aforsyth@lifsci.sdsu.edu Thanks, Pthomas From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!tuli!kenp From: kenp@tuli (Kenneth Prehoda) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 8 Jul 1994 01:16:15 GMT Organization: Division of Information Technology Lines: 122 Message-ID: <2vi9cv$mkf@news.doit.wisc.edu> References: <2vbn6u$j4e@usenet.rpi.edu> <2vcoc2$l6i@news.doit.wisc.edu> <2vdk7s$ss@lyra.csx.cam.ac.uk> <2ve3sq$68q@lyra.csx.cam.ac.uk> <2vejhs$hpd@news.doit.wisc.edu> <2veoju$dur@lyra.csx.cam.ac.uk> <2veuvd$o72@news.doit.wisc.edu> <2vf5cd$i17@lyra.csx.cam.ac.uk> <2vhh69$at6@news.doit.wisc.edu> <2vi071$hv5@lyra.csx.cam.ac.uk> NNTP-Posting-Host: tuli.nmrfam.wisc.edu X-Newsreader: TIN [version 1.2 PL2] Simon Brocklehurst (Bioc) (smb18@mole.bio.cam.ac.uk) wrote: : kenp@banyo (Kenneth Prehoda) writes: : >: They're kinetically inaccessible. That is, if you don't have : >: transition states of sufficiently low energy to allow the protein to : >: pass through them, then you won't get to a particular energy minimum : >: (native state). : >That's my point - they're kinetically inaccessible (in most cases) if : >they are there at all. So of what importance are they? : Sorry, we're agreed on this point! : But I think the pathway from the unfolded to the native state involves : going over energy maxima, and I think this must be where our difference : opinion is (right or not?). : Maybe now's the time to clarify a few points? : 1) Do you believe that when proteins fold they, do so along : particular pathways that are directed by the sequence? : 2) Do you think that these pathways involve traversing energy : maxima (transition states)? : 3) If yes to the transition state idea, do you think that these : states have some native like structure (e.g. partially native : secondary structure)? : 4) Do you think that rather than going through transition states, : there is a roughly downhill path from the unfolded state to the : native state i.e. no significant maxima? : My answers are: : 1) Yes : 2) Yes : : 3) Yes : 4) No : It seems to me that the yes answer to question 3) is particulary : important to arguments about kinetic control? : (stuff deleted) : (talking about side-chain conformations in helices...) : >Well, the researchers in the field that I have talked to believe : >that these restrictions do cause energetic differences which is : >one contribution the the "propensity." : I don't disagree that this is important. It's just that you seemed : to imply that alanine residues are helix forming 'cos they don't make : any unfavourable steric nteractions. : I was just pointing out that some people think otherwise i.e. they : think that alanines intrinsically (i.e. on their own!) like to : be in a helical conformation. (Personally, I don't have strong opinions : on this matter). : (stuff deleted) : >I wish. See Gellman et al. for extensive discussions. My point : >is that the role of hydrogen bonds in protein stability is : >entirely _uncertain_ in contrast to your strong opinion otherwise. : I never meant to imply that hydrogen bonds were not important : contributors to the stability of the native state. : I do believe that the hydrophobic side-chains of residues get : together at least on the same time-scale as H-bonds form, and : quite likely before. Thus I don't see how H-bond formation directs : folding. : >For one thing, how do you measure the strength of a hydrogen bond? : : Tricky do this well: : You can do protein engineering experiments to _try_ to estimate : the strength of hydrogen bonds involving side-chains (see some of : Fersht's work). : You can monitor rates of hydron exhange of amide protons by solution homo : and heteronuclear magnetic resonance spectroscopy. But the : interpretation is complicated by motions other than transient : breaking of hydrogen bonds. This does give a handle on main-chain : H-bonds though. : I'm sure there are loads of ways of doing this, but it's late and : I can't think of any off hand! : Anyway, depending on the conditions the protein is under, the relative : strengths of all kinds of non-covalent interactions will change. : I don't want to flog a dead horse, but I think the important question : about protein folding is: : How does the sequence of the protein limit the number of : conformations that need to be explored to find the native state? : My feeling is that this search involves going uphill as well : as downhill on the free energy surface, traversing highly populated : intermediates and less highly populated transitions states. : Characterising the structures of partly folded states (and even : completely "unfolded" [whatever that means] states) is obviously an : important step forward to understanding protein folding. : Do you really not think that understanding the mechanism(s) by : which proteins fold is interesting/important? : _________________________________________________________________________ : | : | ,_ o Simon M. Brocklehurst, : | / //\, Oxford Centre for Molecular Sciences, : | \>> | Department of Biochemistry, University of Oxford, : | \\, Oxford, UK. : | E-mail: smb@bioch.ox.ac.uk : |________________________________________________________________________ From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Newsgroups: bionet.molbio.proteins Path: biosci!bcm!cs.utexas.edu!sdd.hp.com!saimiri.primate.wisc.edu!news.doit.wisc.edu!psl.wisc.edu!psl.wisc.edu!MCMAHAN From: mcmahan@psl.wisc.edu Subject: Commercial Peptides Message-ID: <1994Jul8.001725.7664@pslu1.psl.wisc.edu> Sender: news@pslu1.psl.wisc.edu (USENET News System) Reply-To: mcmahan@psl.wisc.edu Organization: University of Wisconsin - Physical Science Lab Date: Fri, 8 Jul 94 00:17:25 GMT Lines: 18 I'm thinking about having some peptides made for my research and I was wondering if I could get some feedback from this group about the companies out there. If you have ordered peptides, could you please send the following information: 1) Company name 2) Number of residues 3) Any special side chains, terminus caps etc 4) General impression of service, quality, and speed. 5) Would you order from this company again. I would greatly appreciate any replies I get. Please send them to mcmahan@oncology.wisc.edu Also, if people are interested, I am willing to post a summary of the replies. Thank you in advance for your time and help. Scott McMahan From owner-proteins@net.bio.net Wed Jul 06 23:00:00 1994 Path: biosci!agate!doc.ic.ac.uk!lyra.csx.cam.ac.uk!bjd12 From: bjd12@cus.cam.ac.uk (Ben Davis) Newsgroups: bionet.molbio.proteins Subject: Re: QUESTIONS: alpha-helix "signals" in proteins Date: 7 Jul 1994 10:07:47 GMT Organization: University of Cambridge, England Lines: 65 Distribution: world Message-ID: <2vgk5j$2hv@lyra.csx.cam.ac.uk> References: <2vbn6u$j4e@usenet.rpi.edu> <2ves39$f4f@lyra.csx.cam.ac.uk> <2veuvd$o72@news.doit.wisc.edu> <2vf0o1$pbt@news.doit.wisc.edu> NNTP-Posting-Host: grus.cus.cam.ac.uk X-Newsreader: TIN [version 1.2 PL2] Ken Prehoda (kenp@nmrfam.wisc.edu) wrote: : I have read about this. But to me, this just shows that there is very : little structure in the unfolded state. I mean .2ppm max deviation seems : as strong support for fully unfolded. As far as NOE's I can't remember : any papers describing long range NOE's in an unfolded state. I would : appreciate any references. .2ppm deviation from random coil for a non-labile proton is significant; any structure present is going to be in fast exchange with other (unfolded) conformations, so you can't expect massive deviations. The same idea applies for NOE's - to see a long range NOE, the two protons would have to be in close proximity for a reasonable length of time, which is unlikely in a structure which is (at best) marginal. However, this doesn't mean the protein is unfolded - its not in a random walk conformation - but you do have to look hard for evidence of structure. These structures also tend to be loose associations of hydrophobic sidechains, which (if there's no aromatic residue around) tend to produce only small changes in chemical shift anyway. : > Very good question - can you ever get a totally random coil protein ? I : >suspect under say 6M GuHCl you'd be looking at "totally" denatured, since : >all the aa would be interacting with solvent rather than other aa : >(hopefully). As it is, proteins denatured by acid, by urea, by heat, by : >truncation seem to show residual or marginal elements of structure. : It depends greatly on the protein. Many proteins will not unfold in 6M : GuHCl. True. Just goes to show how hard it is to get a totally random coil ;). I think getting an enviroment where there are no longer *any* interresidue interactions is very hard - that most proteins under denaturing conditions will show some structure (hydrophobic clustering or residual native-like). (I think I just stuck my neck out ...) : > My earlier point was that it'd be good to get an example of a protein : >that *was* totally denatured - just as we need more folded structures, : so we : >need more examples of how proteins behace under denaturing conditions. : Like I said before, I can't imagine a paper where the authors simply : describe a denatured state that has no dispersion. Sounds pretty : boring but maybe that's just me? Can you imagine going through the hassle of assigning a state with no dispersion at all, then saying "well, its boring, lets not publish it after all" ?. Even a negative result is interesting - it's all data. It'd tell you that there was a sequence or conditions where there was *no* residual structure, and since a lot of the folding expts assume they start with a fully denatured state, this is important. : -Ken Prehoda : kenp@nmrfam.wisc.edu Ben _________________________