From owner-7tms_r@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!MAILGATE.GLAXO.COM!true~ta%a1.usa.umc From: true~ta%a1.usa.umc@MAILGATE.GLAXO.COM ("Timothy True") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Streaking, smears and PNGase F Date: 2 Dec 1994 08:47:25 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 43 Sender: daemon@net.bio.net Distribution: world Message-ID: <33645120214991.306670@USA> NNTP-Posting-Host: net.bio.net [This message is converted from WPS-PLUS to ASCII] Barry, I have had success in reducing streaking and the formation of high-molecular weight aggregates/smears when working with photo- affinity labeled GPCRs with the following tricks. (1) Not boiling the samples prior to loading or if you do, minimize the time to 10 to 20 seconds. (2) Don't allow your samples to freeze solid prior to loading (store your samples in 20 to 40% glycerol at -20 C and they should not freeze solid). (3) Store your sample in SDS-PAGE loading buffer overnight in the fridge. With regards to the use of PNGase F (N-Glycosidase F), I have used this enzyme to follow deglycosylation of photo-affinity labelled thromboaxane receptor (D.E. Mais, T.A. True and M. Martinelli, 1992, Characterization by photaffinity labelling of the human thromboxane A2/prostagandin H2 receptor: evidence for N-linked glycosylation, Eur. J. Pharmacol. 227, 267). If you digest your receptor with the endoglycosidase in a batch fashion, and take samples at various time points you can follow the deglycosylation of your receptor (this is pretty cool). The protocol was fairly straight forward. Receptor samples were adjusted to a 5X concentration of the desired digestion buffer, boiled for 10 seconds and cooled on ice. To that I added 4 volumes of high-unit activity endoglycosidase, incubated, took samples, terminated with the addition of SDS-PAGE loading buffer, and stored in the fridge until analysis by SDS- PAGE. For what its worth, I hope this helps. Timothy True Dept. of Cellular Biochemistry Glaxo Reseach Institute TAT32111@Glaxo.com From owner-7tms_r@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!VAX.BIO.LEEDS.AC.UK!BMB6HDD From: BMB6HDD@VAX.BIO.LEEDS.AC.UK Newsgroups: bionet.molbio.proteins.7tms_r Subject: GPCR models and PERSCAN programs Date: 2 Dec 1994 09:23:25 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <20402447_000E2EE8.009885915E1C8820$65_1@UK.AC.LEEDS.BIO.VAX> NNTP-Posting-Host: net.bio.net If anyone who requested my models and/or programs hasn't received anything, then could they re-request them. I had a few problems sending out some of the replies and may well have missed out someone. Dan Donnelly Dept. Biochemistry & Molecular Biology University of Leeds, Leeds LS2 9JT, U.K. bmb6hdd@biovax.leeds.ac.uk Tel. +44 532 33 3117 From owner-7tms_r@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!MRC.COM!huy From: huy@MRC.COM Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: GPCR models available Date: 2 Dec 1994 13:52:21 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <9412022145.AA29065@mrcs1> NNTP-Posting-Host: net.bio.net Dear Dr. Donnelly: Yes, I would be very happy to have a copy of your GPCR models. Please send a copy to: Yinghe Hu Miles, Inc 400 Morgan Lane B24 West Haven, CT 06516 Thank you very much. Yinghe Hu From owner-7tms_r@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!RECEPTOR.PHARM.VIRGINIA.EDU!bg2g From: bg2g@RECEPTOR.PHARM.VIRGINIA.EDU ("Bruce D. Gaylinn") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Using PNGase F with GCRs Date: 2 Dec 1994 07:15:56 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <9412021515.AA06816@receptor.pharm.Virginia.EDU> NNTP-Posting-Host: net.bio.net Sorry, my previous message was cut short. The last line should have read- The enzyme we use is endoglycosidase-F/N-glycosidase-F from Boehringer. -Bruce Gaylinn From owner-7tms_r@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!rutgers!netnews.upenn.edu!pharm15.med.upenn.edu!user From: foisy@pharm.med.upenn.edu (Sylvain Foisy) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Deactivating PNGase F Followup-To: bionet.molbio.proteins.7tms_r Date: 2 Dec 1994 18:59:58 GMT Organization: University of Pennsylvania Lines: 22 Distribution: world Message-ID: NNTP-Posting-Host: pharm15.med.upenn.edu Hi there !! I've been working with receptors and glycasylation and I've been using PHGase F to remove corbohydrate moieities from the receptors. I would like to know if a 90 min incubation at 100 C is sufficent to totally inactivate the enzyme (negative control). Any advice will be appreciated Sylvain Foisy Ph. D. =+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+= Sylvain Foisy | Tel.: (215) 898-9926 Associe de recherche post-doctoral | Dept de Pharmacologie | E-mail: foisy@pharm.med.upenn.edu Ecole de Medecine | Univ. de Pennsylvanie | Philadelphie, PA | 19104-6084 | USA | =+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+= From owner-7tms_r@net.bio.net Thu Dec 01 22:00:00 1994 Path: biosci!RECEPTOR.PHARM.VIRGINIA.EDU!bg2g From: bg2g@RECEPTOR.PHARM.VIRGINIA.EDU ("Bruce D. Gaylinn") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Using PNGase F with GCRs Date: 2 Dec 1994 07:01:22 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <9412021501.AA06780@receptor.pharm.Virginia.EDU> NNTP-Posting-Host: net.bio.net In response to Barry Margulies question, we have been working with deglycosylation of photo-affinity cross-linked GHRH receptor (Endocrinology 135:950-955, 1994). Although in this paper we boiled the samples, we have since gotten even cleaner results with a much simplified protocol. Crude membranes are extracted in 5 mM CHAPS, enzyme is added and incubated at 37 C and deglycosylation is complete in 1 hr. To run on an SDS gel the sample needs to be concentrated and the CHAPS removed. In our system a chloroform- methanol precipitation followed by 37 C incubation in SDS sample buffer works. I have never tried this with anything but GHRH receptor (family 2). The From owner-7tms_r@net.bio.net Sun Dec 04 22:00:00 1994 Path: biosci!chem.gla.ac.uk!chrisw From: chrisw@chem.gla.ac.uk (Chris Wood) Newsgroups: bionet.molbio.proteins.7tms_r Subject: query Date: 5 Dec 1994 12:10:19 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <25008.199412052009@nernst.chem.gla.ac.uk> NNTP-Posting-Host: net.bio.net can you add me to your mailing list so that i can get all the email on GPCRs please chrisw From owner-7tms_r@net.bio.net Mon Dec 05 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!usc!howland.reston.ans.net!pipex!uunet!newstf01.news.aol.com!newsbf01.news.aol.com!not-for-mail From: hmotulsky@aol.com (HMotulsky) Newsgroups: bionet.molbio.proteins.7tms_r Subject: GraphPad Software has a WWW server Date: 6 Dec 1994 09:25:21 -0500 Organization: America Online, Inc. (1-800-827-6364) Lines: 12 Sender: news@newsbf01.news.aol.com Message-ID: <3c1s8h$2ss@newsbf01.news.aol.com> NNTP-Posting-Host: newsbf01.news.aol.com Information on GraphPad Prism is now available on the World Wide Web, along with a demonstration version. Prism is a general-purpose scientific graphing program for Windows, with exceptionally easy curve fitting. It is especially useful for analysis of radioligand binding data and dose-response curves. The WWW server also has information about GraphPad's other programs, InStat (instant statistics, for DOS or Mac) and InTend (laboratory organizer, for DOS). The URL is: http://www.camsci.com/others/graphpad/graphpad.html Dr. Harvey Motulsky, President GraphPad Software HMOTULSKY@AOL.COM From owner-7tms_r@net.bio.net Mon Dec 05 22:00:00 1994 Path: biosci!MIRIS.MED.YALE.EDU!mike From: mike@MIRIS.MED.YALE.EDU ("Michael Singer") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: GraphPad Software has a WWW server Date: 6 Dec 1994 08:00:45 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <9412061102.ZM5466@miris.med.yale.edu> NNTP-Posting-Host: net.bio.net To my knowledge this is first essentially commercial notice posted to this news group. While such notices might be useful and interesting to many in the audience, I am afraid that junk mail will soon overwhelm us. What if every company were to post messages such as this? Perhaps I am be unnecesarily harsh, but is there any policy on this issue? I am interested in comments from other users. Michael Singer Section of Neurobiology Yale School of Medicine From owner-7tms_r@net.bio.net Mon Dec 05 22:00:00 1994 Path: biosci!MODEL.PHR.UTEXAS.EDU!rhodes From: rhodes@MODEL.PHR.UTEXAS.EDU ("David G. Rhodes") Newsgroups: bionet.molbio.proteins.7tms_r Subject: (Fwd) Warning regarding EMail virus Date: 6 Dec 1994 08:59:18 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: world Message-ID: <9412061058.ZM26995@model.phr.utexas.edu> NNTP-Posting-Host: net.bio.net I've heard this from two different sources, so it's possible that these are not just rumors. Anyhow - it's time to forward the info... Warnings are circulating about a virus on Compuserve, America Online, and the like that is being sent by e-mail. The message is apparently captioned "Good Times", and is primarily directed at internet users that use the commercial services for access. The bottom line is, if you get anything called "Good Times", DO NOT READ IT. DO NOT DOWNLOAD IT! IT WILL ERASE YOUR HARD DRIVE! DELETE IT FROM YOUR LIST IMMEDIATELY. ((That's about all I know, so please don't ask me for details.)) Good luck to you all, and please forward the info to your colleagues. Thanks 10^6. -- _____________________________________ O==O _______________________________ | David G. Rhodes | O==O | RHODES@MODEL.PHR.UTEXAS.EDU | | Pharmaceutics Division | O==O | | | College of Pharmacy | O==O | Phone: (512)471-4681 | | The University of Texas at Austin | O==O | Fax: (512)471-7474 | | Austin, TX 78712-1074 | O==O | }:) | |___________________________________| O==O |_____________________________| O==O From owner-7tms_r@net.bio.net Mon Dec 05 22:00:00 1994 Path: biosci!WELCHLINK.WELCH.JHU.EDU!bjmarg From: bjmarg@WELCHLINK.WELCH.JHU.EDU (BARRY J MARGULIES) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Mail Virus (fwd) Date: 6 Dec 1994 12:39:21 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 53 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Now that the original message has circled the globe a few times, and made it to me personally about six times, this disclaimer seems to be in order. Personally, I'm getting a little tired of deleting the "caution, Virus" stuff from my mail. -Barry (bjmarg@welchlink.welch.jhu.edu) xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx GEORGIA CONFEDERATE SOLDIERS from a monument at Antietam National Battlefield, just south We sleep here in obedience to law. of The Cornfield. When duty called, we came. And no, I'm not from Georgia, When country called, we died. I just thought it was cool. xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx ---------- Forwarded message ---------- Sorry. this sort of negates that last warning... -Mark #;?) [Interesting and enlightening quote soon to follow...] ---------- Forwarded message ---------- Date: Mon, 5 Dec 1994 15:28:46 -0500 (EST) From: Suzy Paalman To: Mark Paalman Subject: Re: Mail Virus (fwd) (forwards deleted) From the folks at America Online, paraphrased. This has been circulation fr 3 weeks. No one, to the best of their knowledge, has actually gotten a virus, or trojan horse. It is not possible to put ANSI codes into mail messages, only straight ASCII. It is not possible to do a straight download as in the update program - that only works for AOL-granted updates. It is possible to include a binary in a message, so that it could be downloaded seperately. Their advice - stop sending around messages to everyone, and just be careful like normal. We all have antivirus software, don't we? I personally have received 9 in the last 3 hrs telling me to watch out fro this virus, and have had a bunch of my users come ask me about it. I'm not going to get anything done today, obviously. -- Richard Frueh rvf@netcom.COM -- Just imagine a really neat quote here -- From owner-7tms_r@net.bio.net Mon Dec 05 22:00:00 1994 Path: biosci!rutgers!netnews.upenn.edu!msunews!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!europa.eng.gtefsd.com!emory!usenet From: medtjm@bimcore.emory.edu (T. J. Murphy) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: GraphPad Software has a WWW server Date: 6 Dec 1994 22:29:47 GMT Organization: Biomolecular Computing Resource, Emory University Lines: 31 Distribution: world Message-ID: <3c2okr$a70@emory.mathcs.emory.edu> References: <9412061102.ZM5466@miris.med.yale.edu> Reply-To: medtjm@bimcore.emory.edu NNTP-Posting-Host: bimcore.emory.edu In article ZM5466@miris.med.yale.edu, mike@MIRIS.MED.YALE.EDU ("Michael Singer") writes: >To my knowledge this is first essentially commercial notice posted to this news >group. While such notices might be useful and interesting to many in the >audience, I am afraid that junk mail will soon overwhelm us. What if every >company were to post messages such as this? > >Perhaps I am be unnecesarily harsh, but is there any policy on this issue? I >am interested in comments from other users. > >Michael Singer >Section of Neurobiology >Yale School of Medicine > Given the fact that Harvey's software has made easier the lives of uncounted investigators over the years, I think it's not inappropriate to relax the rules on this one just a tad. It's not like he's getting filthy rich or anything from his hard work. It now means we will have simple access to him for reporting bugs and suggesting improvements. --- TJ Murphy Asst. Professor Dept of Pharmacology Emory University School of Medicine From owner-7tms_r@net.bio.net Tue Dec 06 22:00:00 1994 Path: biosci!rutgers!netnews.upenn.edu!news.cc.swarthmore.edu!psuvax1!news.cac.psu.edu!psuvm!lgm102 Organization: Penn State University Date: Wed, 7 Dec 1994 17:32:18 EST From: Lisa G. May Message-ID: <94341.173218LGM102@psuvm.psu.edu> Newsgroups: bionet.molbio.proteins.7tms_r Subject: subscribe Lines: 1 I would like to subscribe to this newsgruop. From owner-7tms_r@net.bio.net Tue Dec 06 22:00:00 1994 Path: biosci!angis.su.OZ.AU!molgen02 From: molgen02@angis.su.OZ.AU (RPA) Newsgroups: bionet.molbio.proteins.7tms_r Subject: new address Date: 7 Dec 1994 15:34:00 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <199412072331.KAA09660@morgan.angis.su.OZ.AU> NNTP-Posting-Host: net.bio.net my new email address is: mgorrell@med.su.oz.au Mark Gorrell From owner-7tms_r@net.bio.net Wed Dec 07 22:00:00 1994 Path: biosci!dundee.ac.uk!G.G.MCGREGOR From: G.G.MCGREGOR@dundee.ac.uk (Gordon MacGregor) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Reversal of GDPbS bound G proteins? Date: 8 Dec 1994 05:21:48 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <168858D4718@ninewells.dundee.ac.uk> Reply-To: g.g.mcgregor@dundee.ac.uk When using the poorly hydrolyzable analogues of GDP and GTP to study G protein activity is it possible to reverse an inactive GDPbS bound G protein with GTPgS and hence activate the protein, or is the GDPbS bound irreversibly and its removal theoretically and experimentaly impossible? any ideas? g.g.mcgregor@dundee.ac.uk From owner-7tms_r@net.bio.net Thu Dec 08 22:00:00 1994 Path: biosci!PO.CWRU.EDU!pre From: pre@PO.CWRU.EDU (Paul R. Ernsberger) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Commercial announcements Date: 9 Dec 1994 12:33:52 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 39 Sender: daemon@net.bio.net Distribution: world Message-ID: <199412092033.PAA12514@owl.INS.CWRU.Edu> Reply-To: pre@po.CWRU.Edu (Paul R. Ernsberger) NNTP-Posting-Host: net.bio.net > >In article ZM5466@miris.med.yale.edu, mike@MIRIS.MED.YALE.EDU ("Michael Singer") writes: >>To my knowledge this is first essentially commercial notice posted to this news >>group. While such notices might be useful and interesting to many in the >>audience, I am afraid that junk mail will soon overwhelm us. What if every >>company were to post messages such as this? >> >>Perhaps I am be unnecesarily harsh, but is there any policy on this issue? I >>am interested in comments from other users. >> >>Michael Singer >>Yale School of Medicine > >Given the fact that Harvey's software has made easier the lives of uncounted >investigators over the years, I think it's not inappropriate to relax the >rules on this one just a tad. It's not like he's getting filthy rich or >anything from his hard work. > >It now means we will have simple access to him for reporting bugs and suggesting improvements. > >TJ Murphy >Emory University School of Medicine I would reinforce TJ's remarks. Harvey Motulsky is a member of the research community and a number of pharmacologists swear by his software --myself included. He and his products have served an immensely valuable service, but unfortunately GraphPAD is till struggling due mainly to probable piracy. So much for his notice being junk mail. I do think a line should be drawn --I don't want to hear about every new "advance" from Promega by Email unless and independent scientist is recommending it. -- Paul Ernsberger, Ph.D., Depts. of Medicine, Pharmacology and Neuroscience Case Western Reserve University School of Medicine Cleveland, OH 44106-4982 FAX:(216)368-4752 pre@po.cwru.edu Expertise: Receptor Neuropharmacology//Obesity & Hypertension From owner-7tms_r@net.bio.net Thu Dec 08 22:00:00 1994 Path: biosci!WELCHLINK.WELCH.JHU.EDU!bjmarg From: bjmarg@WELCHLINK.WELCH.JHU.EDU (BARRY J MARGULIES) Newsgroups: bionet.molbio.proteins.7tms_r Subject: UL33 N-term (follow up) Date: 9 Dec 1994 10:18:17 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 6 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Sorry 'bout that, folks. I used the wrong keyword to pull out Frank's database from my address book. -Barry From owner-7tms_r@net.bio.net Thu Dec 08 22:00:00 1994 Path: biosci!WELCHLINK.WELCH.JHU.EDU!bjmarg From: bjmarg@WELCHLINK.WELCH.JHU.EDU (BARRY J MARGULIES) Newsgroups: bionet.molbio.proteins.7tms_r Subject: UL33 N-term Date: 9 Dec 1994 09:06:45 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net MTGPLFAIRTTEA From owner-7tms_r@net.bio.net Tue Dec 13 22:00:00 1994 Path: biosci!QUICKMAIL.UCSF.EDU!bruce_conklin From: bruce_conklin@QUICKMAIL.UCSF.EDU ("Bruce Conklin") Newsgroups: bionet.molbio.proteins.7tms_r Subject: Subscribe Date: 14 Dec 1994 13:23:38 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 6 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Subject: Time: 1:19 PM OFFICE MEMO Subscribe Date: 12/14/94 I would like to subscribe to this group, or be on the mailing list. How do I do that? From owner-7tms_r@net.bio.net Sat Dec 17 22:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uunet!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.7tms_r Subject: Q?: high resolution PAGE Date: 16 Dec 1994 19:33:02 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 22 Message-ID: <3csq1e$hj4@sat.ipp-garching.mpg.de> Reply-To: GUIVARC_H@bobby.iaf.cnrs-gif.fr NNTP-Posting-Host: alf.biochem.mpg.de X-Newsreader: TIN [version 1.2 PL2] [ Article crossposted from bionet.molbio.methds-reagnts ] [ Author was GUIVARC_H Dominic ] [ Posted on Fri, 16 Dec 1994 10:23:05 -0200 ] Has anybody had experience about high resolution SDS-PAGE to separate two forms, one ADP-ribosylation-labelled and the other no, of G-proteins. Because I could not resolve the two forms on a normal acrylamide gel. I have an over question about the transfer of the proteins on a nitrocellulose membrane. In the case of the high resolution SDS-PAGE, is there artefacts for the immunoblotting after the separation. Thank you for any suggestions and protocols about this techniques. Best regards. -- G. Dominic GUIVARC_H@bobby.iaf.cnrs-gif.fr Fax 33 1 69 07 05 38 -- /* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */ /* email: krasel@alf.biochem.mpg.de fax: +49 89 8578 3795 */ /* "Science is the game you play with God to find out what His rules are." */ From owner-7tms_r@net.bio.net Sun Dec 18 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!usc!howland.reston.ans.net!agate!usenet From: zeus@mendel.berkeley.edu (Abbas Rizvi) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Crystallograpy Date: 19 Dec 1994 08:46:04 GMT Organization: UC Berkeley Student (USA) Lines: 2 Message-ID: <3d3h8c$er0@agate.berkeley.edu> Reply-To: zeus@mendel.berkeley.edu NNTP-Posting-Host: hi-dive.hip.berkeley.edu X-Newsreader: WinVN 0.92.6+ What's a good book to read about a hands on approach to protein crystal structures? From owner-7tms_r@net.bio.net Sun Dec 18 22:00:00 1994 Path: biosci!VAXA.CRC.MSSM.EDU!STRAHS From: STRAHS@VAXA.CRC.MSSM.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: RE: Crystallograpy Date: 19 Dec 1994 09:50:08 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: <199412191749.JAA02388@net.bio.net> NNTP-Posting-Host: net.bio.net >What's a good book to read about a hands on approach to protein >crystal structures? depend on what you mean by a hands on approach... If you're just interested in protein structure, you might try Creighton's "Proteins" or the Branden and Tooze text "Introduction to Protein Structure".. alternatively, if you're more interested in the hows and whats of crystallography, you might look at the International Union of Crystallography publications, such as the IuCr Monographs and IuCr Crystallographic Symposia. especially, you probably want to look at the IuCr Texts on Crystallography; in particular, volume 2 "Fundamentals of crystallogrphy", Giacovazzi, ed. The Cantor and Schimmel texts "Biophysical Chemistry" also discuss crystallography in some detail, in volume 2, if I remember correctly. If you're looking for the "Big Book of How Molecular Biologists can grow nice crystals of integral membrane proteins", than I suspect you're wasting your time and it would be much more time-efficient for you to approach your local structural person (xtal/NMR) and talk to them directly. Crystal growing is a problem that has been somewhat redacted, but it is still very much a difficult problem. From owner-7tms_r@net.bio.net Mon Dec 19 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!sgiblab!swrinde!howland.reston.ans.net!agate!msunews!netnews.upenn.edu!brass2.med.upenn.edu!user From: obrien@pharm.med.upenn.edu (PJOB) Newsgroups: bionet.molbio.proteins.7tms_r Subject: genomic GPCR sequences Followup-To: bionet.molbio.proteins.7tms_r Date: Tue, 20 Dec 1994 11:54:19 -0500 Organization: University of Pennsylvania Lines: 15 Message-ID: NNTP-Posting-Host: brass2.med.upenn.edu Many apologies in advance if this is a FAQ... I am trying to determine which GPCRs have been characterized at the genomic DNA level. Specifically, I am interested in intron/exon boundaries in ORFs. My medline searches have yielded very few hits, and the Web site for this group wasn't much help... Any suggestions (ie. references) will be greatly appreciated. Peter O'Brien Univ. of Pennsylvania obrien@pharm.med.upenn.edu -- Opinions seen here are channeled to me by a Ring-Tailed Lemur named Oxnyx From owner-7tms_r@net.bio.net Wed Dec 21 22:00:00 1994 Newsgroups: bionet.molbio.proteins.7tms_r Path: biosci!agate!howland.reston.ans.net!Germany.EU.net!news.dfn.de!scsing.switch.ch!rzusuntk.unizh.ch!NewsWatcher!user From: maga@vetbio.unizh.ch (Giovanni Maga) Subject: Re: Q?: high resolution PAGE Message-ID: Followup-To: bionet.molbio.proteins.7tms_r Sender: newsadm@rzu-news.unizh.ch (CNEWS ADMINISTRATION) Organization: University of Zurich Irchel- Biochemistry References: <3csq1e$hj4@sat.ipp-garching.mpg.de> Date: Thu, 22 Dec 1994 11:47:39 GMT Lines: 38 In article <3csq1e$hj4@sat.ipp-garching.mpg.de>, krasel@alf.biochem.mpg.de (Cornelius Krasel) wrote: > [ Article crossposted from bionet.molbio.methds-reagnts ] > [ Author was GUIVARC_H Dominic ] > [ Posted on Fri, 16 Dec 1994 10:23:05 -0200 ] > > Has anybody had experience about high resolution SDS-PAGE to separate > two forms, one ADP-ribosylation-labelled and the other no, of G-proteins. > Because I could not resolve the two forms on a normal acrylamide gel. I > have an over question about the transfer of the proteins on a > nitrocellulose membrane. In the case of the high resolution SDS-PAGE, is > there artefacts for the immunoblotting after the separation. > Thank you for any suggestions and protocols about this techniques. > Best regards. > > -- > G. Dominic > GUIVARC_H@bobby.iaf.cnrs-gif.fr > Fax 33 1 69 07 05 38 > > -- > /* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */ > /* email: krasel@alf.biochem.mpg.de fax: +49 89 8578 3795 */ > /* "Science is the game you play with God to find out what His rules are." */ Re: G. Maga Uni-Irchel, Zh (CH) Biochemistry E-mail: maga@vetbio.unizh.ch I have no direct experience with G-proteins, but I think you could get a better resolution by using a gradient PAGE. Biorad is selling precasted gradient gels, but you can make your own gradient (optimized for your protein) simply by loading a preformed PA gradient in a normal gel apparatus (principally like glycerol gradients). This could be the simpliest way. Other tecniques that could help you are IEF, 2D gels, but I guess they are more time-consuming. Kind regards, Giovanni Maga From owner-7tms_r@net.bio.net Thu Dec 22 22:00:00 1994 Path: biosci!CS.Arizona.EDU!uunet!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: Nice serpent pictures Date: 23 Dec 1994 17:27:30 GMT Organization: University of Michigan Lines: 12 Message-ID: <3df1a2$5sc@lastactionhero.rs.itd.umich.edu> NNTP-Posting-Host: warbler.med.umich.edu I know this is not the most intellectually stimulating question but: How do people make those nice pictures of 7TM's with all of the aminoacids labelled in little circles to show the membrane spanning regions and loops? Is there some software package that does that or is it just a standard drawing package with lots of hard manual work to make them look right? Happy holidays! Rick Neubig http://www.umich.edu/~rneubig From owner-7tms_r@net.bio.net Fri Dec 23 22:00:00 1994 Path: biosci!MSCF.MED.UPENN.EDU!brass From: brass@MSCF.MED.UPENN.EDU (Skip Brass) Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Nice serpent pictures Date: 24 Dec 1994 09:04:22 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 31 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HL0ROYZVAA005KV4@mail.med.upenn.edu> NNTP-Posting-Host: net.bio.net At 5:27 PM 12/23/94 +0000, Rick Neubig wrote: >I know this is not the most intellectually stimulating question but: > >How do people make those nice pictures of 7TM's with all of the >aminoacids labelled in little circles to show the membrane spanning >regions and loops? Is there some software package that does that or is it >just a standard drawing package with lots of hard manual work to >make them look right? > >Happy holidays! > >Rick Neubig >http://www.umich.edu/~rneubig Rick: Blood, Sweat and Fears. Skip ********************** Skip Brass University of Pennsylvania brass@mail.med.upenn.edu 215-573-3540 phone 215-573-2189 fax ********************** From owner-7tms_r@net.bio.net Fri Dec 23 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Nice serpent pictures Date: 25 Dec 1994 03:25:37 GMT Organization: University of Michigan Lines: 23 Message-ID: <3dionh$gmi@lastactionhero.rs.itd.umich.edu> References: <01HL0ROYZVAA005KV4@mail.med.upenn.edu> NNTP-Posting-Host: pm002-12.dialip.mich.net brass@MSCF.MED.UPENN.EDU (Skip Brass) wrote: > >How do people make those nice pictures of 7TM's with all of the > >aminoacids labelled in little circles to show the membrane spanning > >regions and loops? > > Rick: > > Blood, Sweat and Fears. > > Skip > Oh well. I had hoped for a more elegant (and less painful) solution. ************************************************************** -------------------------------------------------------------- Rick Neubig http://www.umich.edu/~rneubig -------------------------------------------------------------- ************************************************************** From owner-7tms_r@net.bio.net Sun Dec 25 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!uhog.mit.edu!europa.eng.gtefsd.com!newsxfer.itd.umich.edu!news.itd.umich.edu!usenet From: Rick Neubig Newsgroups: bionet.molbio.proteins.7tms_r Subject: Re: Nice serpent pictures Date: 26 Dec 1994 18:51:37 GMT Organization: University of Michigan Lines: 20 Message-ID: <3dn3bp$q6g@lastactionhero.rs.itd.umich.edu> References: <01HL0ROYZVAA005KV4@mail.med.upenn.edu> <3dionh$gmi@lastactionhero.rs.itd.umich.edu> NNTP-Posting-Host: pm002-04.dialip.mich.net Rick Neubig wrote: > > > >How do people make those nice pictures of 7TM's with all of the > > >aminoacids labelled in little circles to show the membrane spanning > > >regions and loops? I got several other answers to this question via direct e-mail and the universal answer was: We make them by hand (ususally from a draw file that has been handed down from generation to generation of students and postdocs). Oh well. I guess that's the state of the art! Thanks for the info to all who wrote. ---------------------------------------------------- Rick Neubig http://www.umich.edu/~rneubig From owner-7tms_r@net.bio.net Mon Dec 26 22:00:00 1994 Path: biosci!SHRSYS.HSLC.ORG!wthomas From: wthomas@SHRSYS.HSLC.ORG Newsgroups: bionet.molbio.proteins.7tms_r Subject: carboxy-terminal tails Date: 27 Dec 1994 15:23:42 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <0098993F.6A7ECE13.6@shrsys.hslc.org> NNTP-Posting-Host: net.bio.net Hi to all! From owner-7tms_r@net.bio.net Mon Dec 26 22:00:00 1994 Path: biosci!SHRSYS.HSLC.ORG!wthomas From: wthomas@SHRSYS.HSLC.ORG Newsgroups: bionet.molbio.proteins.7tms_r Subject: carboxy-terminal structure Date: 27 Dec 1994 15:44:54 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <00989942.6184B77F.5@shrsys.hslc.org> NNTP-Posting-Host: net.bio.net I'm interested in finding out the secondary structure of the c-terminal tail of the angiotensin II (type AT1A) receptor. While I realize that the tails of GPCRs are of variable lengths, is there any consensus as to the secondary structure(s) they adopt? I recall reading somewhere that they may be loosely structured and simple "float" in the From owner-7tms_r@net.bio.net Mon Dec 26 22:00:00 1994 Path: biosci!SHRSYS.HSLC.ORG!wthomas From: wthomas@SHRSYS.HSLC.ORG Newsgroups: bionet.molbio.proteins.7tms_r Subject: carboxy-tails Date: 27 Dec 1994 16:08:39 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 25 Sender: daemon@net.bio.net Distribution: world Message-ID: <00989945.B36334D3.6@shrsys.hslc.org> NNTP-Posting-Host: net.bio.net Sorry to those who received two previous incomplete mailings. My full query is: I'm interested in finding out the secondary structure of the C-terminal tail of the angiotensin II (type AT1A) receptor. While I realize that the tails of GPCRs are of variable length, is there any consensus as to the secondary structure(s) they adopt? Are they loosely structured, simply "floating" in the cytoplasm or are they predicted to form tight coils and turns? Also, are there any PC based programs available which can predict such structures? Just in case a protein chemist is reading, the amino acid sequence for the AT1A receptor tail is: NPLFYGFLGKKFKKYFLQLLKYIPPKAKSHSSLSTKMSTLSYRPSDNMSSSAKKPASCFEVE Hope someone can help. Thanks, Wally Thomas Weis Center for Research, Geisinger Clinic, Danvill, PA e-mail:wthomas@shrsys.hslc.org From owner-7tms_r@net.bio.net Wed Dec 28 22:00:00 1994 Path: biosci!VAXA.CRC.MSSM.EDU!STRAHS From: STRAHS@VAXA.CRC.MSSM.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: RE: carboxy-tails Date: 29 Dec 1994 07:48:12 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 76 Sender: daemon@net.bio.net Distribution: world Message-ID: <199412291548.HAA08449@net.bio.net> NNTP-Posting-Host: net.bio.net From: MX%"STRAHS@VAXA.CRC.MSSM.EDU" 28-DEC-1994 12:21:51.03 To: MX%"wthomas@shrsys.hslc.org" CC: MX%"7tms_r@net.bio.net" Subject: RE: carboxy-tails >I'm interested in finding out the secondary structure of the C-terminal tail >of the angiotensin II (type AT1A) receptor. > >While I realize that the tails of GPCRs are of variable length, is there any >consensus as to the secondary structure(s) they adopt? Are they loosely >structured, simply "floating" in the cytoplasm or are they predicted to >form tight coils and turns? Also, are there any PC based programs >available which can predict such structures? > >Just in case a protein chemist is reading, the amino acid sequence for the >AT1A receptor tail is: > >NPLFYGFLGKKFKKYFLQLLKYIPPKAKSHSSLSTKMSTLSYRPSDNMSSSAKKPASCFEVE > >Hope someone can help. Thanks, > >Wally Thomas >Weis Center for Research, >Geisinger Clinic, >Danvill, PA >e-mail:wthomas@shrsys.hslc.org First, there is no consensus as to any possible structure. Not only are the tails of variable length (as you mentioned), but there is little or no conservation among the receptors. Yes, all Delta Opiate receptors have roughly the same sequence, but the Mu Opiates and the Kappa Opiates already diverge in both sequence and length. There are several different Angiotension receptor tail ends. You listed one above; here are several others: RATAGT2A GKKFKKYFLQLLKYIPPTAKSHAGLSTKMSTLSYRPSDNMSSSAKKSASFFEVE RATAGT2B GKKFKKYFLQLLKYIPPKAKSHSSLSTKMSTLSYRPSDNMSSSAKKPASCFEVE RATAGT2C GNRFQQKLRSVFRVPITWLQGKRETMSCRKSSSLREMDTFVS MUSAGT2A GKKFKKYFLQLLKYIPPKAKSHSSLSTKMSTLSYRPSDNMSSAAKKPASCSEVE MUSAGT2B GKKFKRYFLQLLKYIPPKARSHAGLSTKMSTLSYRPSDNMSSSARKSAYCFEVE MUSAGT2C GNRFQQKLRSVFRVPITWLQGKRETMSCRKGSSLREMDTFVS HUMAGT2 GKKFKRYFLQLLKYIPPKAKSHSNLSTKMSTLSYRPSDNVSSSTKKPAPCFEVE PIGAGT2 GKKFKKYFLQLLKYIPPKAKSHSSLSTKMSTLSYRPSENGSSSTKKSAPCTEVE BOVAGT2 GKKFKKYFLQLLKYIPPKAKSHSNLSTKMSTLSYRPSENGNSSTKKPAPCIEVE RABAGT2 GKKFKKYFLQLLKYIPPKAKSHSNLSTKMSTLSYRPSDNVSSSSKKPVPCFEVE TKYAGT2 GKNFKKYFLQLIKYIPPNVSTHPSLTTKMSSLSYRPPENIRLPTKKTAGSFDTE FRGAGT2A GKNFRKHFLQLIKYIPPKMRTHASVNTKSSLVSSSLSDTKRASKKIALQMTDNEEHCK FRGAGT2B GKKFRKHFLQLIKYIPPKMRTHASVNTKSSTVSQRLSDTKCASNKIALWIFDIEEHCK Not only is there no consensus as to what the structure of these regions are, but there is no consensus as to what this region is. For example, I believe your definition of the region above is incorrect. The asparagine of the "NPxxY" motif in helix 7 has been shown to interact with the conserved aspartate in helix 2 (Zhou et al. (1994) Mol. Pharm. 45(2), 165-170). While the Lefkowitz's lab interpretation of the conserved tyrosine in this motif as required for sequestration is probably correct, it is not required that it be in the cytoplasm. If you want to predict the structure of this region, go ahead. Plenty of programs will do what you want (such as Macvector, DNAStar, the GCG package, the Rost/Sander package at EMBL, etc.). Whether any of these programs will get it correct is another matter entirely, and the answer is probably no. The best secondary structure prediction program is about 70% accurate at this point, with little forseeable improvement for some time. Your best bet may be to collaborate with one of the biophysical labs that models the angiotensin GPCR's. For example, there was a recent JBC paper published with Harold Scheraga. Alternatively, maybe you want to try to crystallize this domain, or explore it with NMR or CD or some such technique. Other than all this, good luck. Dan Strahs Physiology and Biophysics Mt. Sinai School of Medicine From owner-7tms_r@net.bio.net Thu Dec 29 22:00:00 1994 Path: biosci!SMTP.GEISINGER.EDU!KBAKER From: KBAKER@SMTP.GEISINGER.EDU Newsgroups: bionet.molbio.proteins.7tms_r Subject: g-protein receptor coupling to tyrosine kinase events Date: 30 Dec 1994 10:01:43 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net We have recently shown coupling of the AT1 angiotensin II receptor to activation of a number of tyrosine kinase related events. These include Shc, Fak, and Stat proteins. Does anyone have information as to how G-protein receptors in general couple to these events? K.M. Baker Weis Center for Research kbaker@SMPT.Geisinger.edu