GFP meeting summary (11 pages)

ichida at BIOMAIL.UCSD.EDU ichida at BIOMAIL.UCSD.EDU
Fri Oct 31 17:46:16 EST 1997


Hello GFPers,

This long message contains a summary of the recent GFP meeting. It is
completely unoffical. I was not asked to do this by the organizers, nor
have they seen it (except Daniel Gonzalez). This was just a little idea I
had about sharing the excitement with all of you. 11 meeting attendees
ended up contributing to the content. Between all of our notes we managed
to put together this outline. It turned out to be much longer than I
originally thought it would be, but as a result it should be that much more
useful. Some of the summaries are short and others are long. This happened
because the 11 contributers were just nice people I met at the meeting and
they were not chosen for optimum coverage of all topics. 

Yikes 11 pages! But we all sure had a lot of fun putting it together. I
hope you have fun reading it.

Sincerely,
Audrey Ichida

GFP MEETING SUMMARY

The International Symposium on GFP was sponsored by Rutgers University, New
Brunswick, New Jersey Oct. 18-22, 1997 (Rumor has it the next meeting will
be spring of 1999.) http://www.rci.rutgers.edu/~meton/GFP_Symposium.html

We would like to thank the meeting organizers for a job well done. They
volunteered their time and energy.  Drop them a thank you note if you
enjoyed the meeting!
Bill Ward
Daniel Gonzalez
David Piston
Clifford Mintz
James Slock
Douglas Prasher
Also check out the web site for links to corporate sponsors. They sent
their best and brightest to manage the booths and this was much
appreciated.

disclaimer: This is an informal account of the many interesting talks that
were presented at the GFP meeting. Please note that most speakers were not
consulted about the contents of the presented summaries, so this
information is hearsay. This is meant as a sound-bite summary and is only
meant to whet your appetite and generate discussion within this group. Also
no one has been paid to do this so it is NOT an advertisement or
endorsement.  We encourage meeting attendees and speakers to correct any
errors that may be present in this summary. We felt that timeliness was
more important than waiting to get proof-reading from each speaker. The
internet is supposed to be self correcting, so stay tuned to fluorpro.

Chronologically listed
SATURDAY 

MARTIN CHALFIE
- presented his connection to GFP, first cloning etc.

D.D. MOSSER
- use of GFP in retroviral vectors to monitor gene expression in
tet-inducible and heat shock inducible constructs.
- use of GFP in E1 -region of Adenovirus to monitor adenovirus mediated
DNA-transfer

DAVID DORSKY
- presented use of GFP to monitor HIV-infections with a LTR-GFP-construct

FRANK PETERSON
- Development of an ELISA to detect low amounts of GFP ( down to pg)

DEBRA S. GREGA
- Development of monoclonal Antibodies for WB and/or IP

RAINER KOHLER   (Cornell University) gave two talks
-presented mitochodrial targeted GFP in tobacco and petunia
-also imaged tobacco trichomes, showed movies of moving mitochondria and
still choloroplasts 
-presented GFP targeted to chloroplast stroma
-has re-discovered a structure he has named stromules, These are tubules
that stick out of cholorplasts and also connect cholorplasts (see Science
June 27, 1997 and Trends in Cell Biology Oct.. 1997, he says there are
movies on his web site)
-Physical connection confirmed by FRAP.

BEAT LUDIN (Friedrich Miescher Institut) (Beat is pronounced Bee-at)
-showed dynamics of tau-GFP and MAP2-GFP-decorated microtubules in 
fibroblasts
-showed microtubule and actin dynamics using beta tubulin-GFP, gamma 
cytoplasmic actin-GFP and GFP-actin
-movies up to 6 hours time lapse
- showed morphological plasticity of dendritic spines in mature 
hippocampal neurons visualized by GFP actin. Plasticity is inhibited by 
actin poisons (cytochalasins, latrunculin)
-GFP-actin + MAP2c-BFP co-imaged show independent movement of microtubule 
bundles and stress fibres in fibroblasts
(-in partnership with Novartis for development of drug screening methods 
and for apoptosis research)
-movies on web site www.fmi.ch/groups/AndrewMatus/Group.html
Q&A: use culture media with low riboflavin for lower background

SUNDAY

JIM MORIN (Cornell University)
-co-localize bioluminescence and fluorescence in Cnidarians
- bioluminiscence is used mainly for defense, communication and obtaining 
food in 100%, 70% and 40% respectively of the species investigated.
- visual abilities of fish best in the green range - one possible reason 
for the evolution of GFP

JOHN WAMPLER (U. of Georgia) talked 3 times 
- GFP enhances the quantum efficiency of aequorin due to non-radiative 
energy transfer.
-co-localize bioluminescence and fluorescence in Renilla
-noted diversity of green:blue light ratio in different colonies of Renilla
mulleri
-isoelectric focusing to band GFP back in 1971 (historical note)

WOODY HASTINGS (Harvard)
-looks at many different genera and compares spectra
-about 30 different systems have been described to have biofluorescence
only 7 different substrates and enzymes identified
-possibly independent evolutionary origins? This is a future question.

TONY CAMPBELL (U of Wales)
-showed slides of deep sea red-emitting organisms
- fused aequorin to GFP to make a brighter Ca2+ indicator (manuscript in
preparation)
- Aequorin-GFP-fusions with Kinase site in between for FRET
- half life of aequorin in cytoplasm is short (20')
- mentioned high-sensitivity imaging by directly coupling of 
fiber-optics to coverslips. 

BILL WARD (Rutgers)
-described heroic collection and purification efforts involved in GFP
isolation pre-cloning
-a month of collection (8 people putting in 12 hour days = 100,000
jellyfish) plus 6 months of purification = 200-500 mg of pure GFP
-knows how to separate dimer from monomer

BRIAN HERMAN (UNC-CH)
-FRET between BFP-Bcl2 and GFP-Bax
-looked at the apoptotic pathway by looking at the time course of
mitochondrial membrane potential loss and the interactions of Bcl2 and Bax
- release of GFP-tagged cytochrome C from mitochondria during apoptosis

OLE THASTRUP (BioImage/Novo Nordisk)
-he mentioned kinase sites in GFP, but did not present data
- looked at the redistribution and GFP kinase fusions to monitor the kinase
activity and levels of cAMP.
-GFP fusions to PKC alpha, PKA and Glut4
-Glut4 localizes to large, outward moving structures in insulin stimulated
CHO. (I think it was punctate w/o stimulation, not sure though.)
Co-localizes with actin. 
-PKC-GFP evenly distributed after carbachol it goes to PM within seconds
-PMA phorbol ester slow and irreversible, carbachol fast and reversible
-PKA-GFP punctate then added cAMP(-inducers like forskolin) and saw even
distribution 
-In contrast, GFP-PKA always remains evenly distributed 

PETER MOMBAERTS (Rockefeller)
-showed beautiful 3D spinning neurons from mouse heads
-mouse "knock-ins", add genes
-GFP fusion to olfactory receptors (OMP-tau-GFP and P2-IRES-tau-GFP, also
has these fusions with lacZ)
-showed that olfactory neurons express a single receptor (out of 1000). 
-all neurons with identical receptors project to only two glomeruli on the
average
- in-tissue imaging approaches single axon-resolution using 2-photon 
confocal microscopy

ROBERT HOFFMAN (Anticancer Inc. San Diego, together with Yokohama City 
University School of Medicine, Japan)
-orthotopic transplant of human cancers that have been GFP labeled into
mice (nude)
-has done this with colon, prostate and kidney, will try with breast cancer)
-watches cellular level metastasis in living tissue and histoculture

EUGENE KANDEL
-Use of GFP in retroviral constructs with GFP as marker for transfection
for FACS
-He discussed briefly the recent paper implying GFP toxicity in mammalian cells
-retroviral vectors with different structures of transcriptional units were
designed  
-notes that constructs commonly used for highest GFP expression are
suboptimal for coexpression of another gene. 
-collects cells based on fluorescence 2 days post infection so can study
effects of the proteins for which long-term expression is impossible. 
-observed no detrimental effect of GFP in very sensitive long-term studies
-can specifically select cells with high copy number of integrated
proviruses and separate on the same fluorescence plot 3 populations:
negative, S65T -  and EGFP- expressing  (saves you from using other markers
or buying UV laser for your FACS). 
-placed GFP in an antisense towards regulated promoter: can see regulation
by decrease or increase in fluorescence, but do not have to turn the
regulated promoter "on" to detect
gene transfer (good for working with "toxic" genes).


ANDREAS BUPERT (Biocenter U. Wurzberg, Germany)
-GFP as a promoter reporter for virulence genes (mammalian)
done with Listeria monocytogenes, GFP under control of virulence gene
promoters

ATSUSHI MIYAWAKI (UCSD)
-stressed potential unique features of FRET with GFP, compared with the
ordinary FRET with small fluophores.  FRET with GFP is not a good
spectroscopic ruler, but it is a good detector for protein conformational
change!  Relative orientation of two dipoles should give a significant FRET
change.
-cameleons (see Aug 28th Nature)
-BFP/CFP-CaM-2glycines-M13(CaM binding peptide)-GFP/YFP as Ca2+ indicators
(uses intramolecular FRET)
-to optimize FRET change, many linker sequences (> 30) were tried;
sometimes only one amino acid ins/del or exchange drastically changed the
FRET, suggesting some contribution of orientation factor.
-cameleons are fluorescent Ca2+ indicators which are entirely encoded by
cDNAs, easily targetable to any sites, and single cell imaging is possible.
Q&A: noise problem, still significant FRET in absence of Ca2+
        -also as is standard they don't really work in low pH (<7)

NEAL CARIELLO (Glaxo Wellcome Inc.)
-GFP for mutagen assay in bacteria
-back mutation, CCCC to CCC to gain GFP function, resembles only frame
shift mutagens
-even with ~200 copies of GFP plasmid per cell, one copy mutated is
sufficient to see GFP
-forward mutation, used Arabinose operon, in presence of glucose GFP
transcription is repressed unless the repressor, promoter or GFP is mutated
with substitution mutagens
-forward assay is more sensitive against point mutations, but insensitive
against frameshift mutagens

MARK PRESCOTT (Monash U., Australia)
-fused GFP to different components of ATP synthase complex in yeast mito
-OCSP-GFP used to monitor turnover of complexes and response to stimuli
-plans to use BFP and S65T in one cell to look at FRET within complex

MATTHIAS VOGAL (Universitat Regensburg)
-Epstein-Barr virus OriP, GFP as operon reporter
-FACsort cells

EINAR HALLBERG (Stockholm U.)
-GFP fusions to nuclear pore proteins (nucleocytoplasmatic site of POM121
has a nucleopore targeting signal)
-looked at pore distribution, plan to look at assembly and disassembly in
mitosis
-GFP tagged deletion mutants to look for targeting signals
-neuroblastoma cells, plan to look at nuclear pore division
-able to monitor loss of nuclear pores as an early marker of apoptosis
before DNA fragmentation
- loss of nuclear pores could be used to distinguish apoptosis from necrosis

JOHN RASKO (Fred Hutchinson Cancer Research Center)
-has 3 stable GFP-retrovirus production lines
-advantages over other cyotchemical techniques or drug resistance in terms
of time, cost.
-sometimes 1/2 a colony has GFP switched off, interprets this as indication
of GFP cytotoxicity
-some attempted packaging cell lines would not produce stable GFP expression 
-has some better packaging cell lines

CHRISTER LINDQVIST (Abo Akademi U., Finland)
-Baculovirus expression of GFP for FACS of transfected cells
-hydroxylapatite column can be loaded with Zn2+ (or Ni2+ or Cu2+) and then
be used for metal affinity chromatography of His-tagged GFP (with moderate
success)

MONDAY

JOHN ANDREWS (U Wisconsin)
-GFP tagged cells introduced to microbial population to watch interactions
and measure population dynamics easily without taking them out of their
environment
-uses TEF (translation elongation factor) promotor for constitutive construct
-Uses Xylanase promoter for inducible construct
-impact of GFP-tagging on generation time is marginal (wt 4.6 h, + GFP 5.2 h)

JANET JANSSON (Stockholm U.)
- detects GFP-expressing bacteria in soil samples as a method for
monitoring the spread of (genetically manipulated) microorganisms.
-screened 100s of transformants to find one that could grow on chlorophenol
-heat kill bacteria and loose GFP as test for live cell assay (based on
propidium iodide), in which it
should be determined if GFP expressing cells are still alive
-fluorescence loss after heat treatment (50 C ?) is probably due to
diffusion of GFP out of the cell (cells get porous)
-organism is Arthrobacter sp A-6
-GFP fluorescence is stable upon starvation (N, C)
-luciferase activity vanishes upon starvation

NEAL STEWART (U. North Carolina)
-plants expressing mGFP5 (in ER) planted in field studies 
-use portable equipment for detection of GFP marked plants (walk in the
field at night)
-in future hope to use different shifted GFPs to tag different resistance
genes to follow out-crossing into wild weedy relatives
-GFP expression is negligible cost to overall plant growth (reproduction
not looked at yet)
-chlorophyll masking might be a problem
-when a leaf is growing shaded by another leaf, the shaded leaf has much
less GFP fluorescence. 
(the shade leaf concept was covered by Dr. Galbraith during the 
questions.  Theoretically, the shade induced more chlorophyll 
production which masked the GFP.  Other people at the meeting found 
that etiolated plants were the best way to view GFP in planta without 
chlorophyll interference. )www.uncg.edu/~cnstewar/

CLAUS STERNBERG (Techn. U. Denmark)
-in P. pudita, express GFPs with degradation signals
- uses TSPD(Science 271:990) to destabilize GFP to use it as a marker for
promoter activity ...LAA leads to protein (and fluorescence) half life time
of 30 min upon shift to minimal medium (without C-source)
- to test it, used it with a ribosomal promoter that should only be active
in pre-stationary phase, means proliferating bacteria (worked)
  
STEVE KAIN (Clontech)
-history of Clontech's EGFP and it's properties
-made a dEGFP which is a better RNA reporter because it is destabilized
with a half-life of ~2 hrs as opposed to >>24h for EGFP 
-precise truncation studies show residues 7-229 to be the minimal construct
Q&A: Bill Ward said pure GFP in azide in fridge still glows after 20 years

PAUL KITTS (Clontech)
-showed EBFP and EYFP spectra
- discussed which mutations causes which effects in their GFP constructs
-data for mutants:
 *EBFP F64L/Y66H/S65T/Y145F, humanized codon usage, Ex 380 nm, Em 440 nm,
epsilon = 31000, quantum yield 0.2
 *EYFP S65T/V68L/S72A/T203Y Ex 513 nm, Em 527 nm, epsilon = 36500, quantum 
yield 0.63
-it is possible to separate EGFP and EYFP by FACS (Ex 488 nm, 
measured at 507/523 nm resp.)

ANDREW CUBITT (Aurora Biosciences Corp.) (Company formed by Roger Tsien,
Charles Zuker and Geof Rosenfeld)
-note that upcoming review will detail 20 different mutants
-gave specs on Topaz, Emerald and Sapphire GFPs, distributed by PACKARD
(http://www.packardinst.com) 
*Emerald F64L,S65T,S72A,N149K,M153T,I167T,H231L, 484 nm excite, 508 nm emit
*Topaz S65G,S72A,K79R,T203Y,H231L, 514 nm excite, 527 nm emit
*Sapphire S72A,Y145F,T203I,H231L, 395 nm excite, 511 nm emit
-some of the new mutations are extremely pH sensitive up to 7.5 so that old
measurements are off.
(epsilons up to 94000 !)
-has a FRET based protease test, after FRET pair optimization the change in
emission peak ratio (507 nm/450 nm) is up to 5.2
-small stokes shift seemed to work best for FRET donors
--has phosphorylation sensitive GFP (for PKA, PKC and ERK1) that shows rapid 
kinetics and 5-7x change upon phosphorylation (dynamic range).  KM in the
micromolar range so not as good as peptides but assays are nonradioactive
and can be run at either high or low ATP concentrations, validated by
inhibitors, however not reversible

GEORGE PAVLAKIS (NCI-FCRDC)
-showed structure of blue mutant
-has GFP tagged HIV proteins (Gag, Nef, Rev, Tat and Vpr)
-tag receptors with GFP and co-loc with fluorescent ligand
-notes that antibody does not always completely agree with GFP because
permeablization can loose cytoplasmic GFP
-www.pavlakislab.ncifcrf.gov
-thinks EGFP may work better because less AT content stabilizes GFP, and
not so much the codon usage, developed enhanced blue and enhanced green
mutant without CODON optimization (available from QUANTUM)

MARKUS MANIAK (Max-Planck-Institut fur Biochemie)
-GFP in dicty
-GFP-actin and GFP-coronin
-Proton-ATPase-GFP 
-endocytosis of media with TRITC-dextran and watched change from pH 7-10 in
10 min.
-also FITC-dextran that bleaches in an acidic environment (gives red(TRITC)
instead of yellow(FITC+TRITC) in the endosomes)
-Vacuolin-GFP loc in a bright dot that is assoc. with a faint ring that is
vacuole, treat with DMSO and vacuolin-GFP outlines the vacuole
-GFP-tubulin to look at role of MTs in endocytosis
-also have GFP-actin6 in cell which can also run on myosin heads in vitro

HANS-HERMANN GERDES (U of Heidelberg)
-applied a 20 degree block to accumulate in the TGN, then released the
block and observed random vesicle movement, and secretion on a Ca-signal
-GFP fusion to human chromogranin B
-secretory granules leave transgolgi network and go to PM
-movies of vesicles, they show stochastic retrograde and anterograde
movement on microtubules and they also may leave microtubules, speed was
1micron / sec (at 37 degrees 3-4micron/sec)
-showed that the target membrane was more important that the direction of
movement for ultimate targeting.(microtubule-based transport enhances speed
but not directionality)
- the GFP probe is a better transport indicator than acridine orange.

JOHN LEVY (Human Gene Therapy Research Institute)
-retroviral transfer of GFP for identification and sort of positive clones
and transfected cells in tumors
- mentioned that initially the development of humanized GFP was actually a
by-product/control for another experiment

MONDAY NIGHT --- GREEN FOOD AND GLOW STICKS!!!

TUESDAY

SHARYN ENDOW (Duke U.)
-Drosophila oocytes NCD-GFP (minus-end directed kinesin) also gamma-tubulin-GFP
-showed movies of photobleach spindle and recovery during meiosis and mitosis
-Ncd-GFP is present across the metaphase plate in contrast to microtubule
immunostaining 

JENNIFER MOSS (Massachusetts General Hospital)
-muscle specific GFP in zebra fish and mouse
-GFP under Myosin light chain promoter
-has GFP in fast and slow fibers

DAVID GALBRAITH (U Arizona)
-flow cytometry of maize or tobacco protoplasts
-had to make the GFP construct >60 Kd  to prevent GFP in both the nucleus
and cytoplasm;  in smaller constructs GFP in cytoplasm goes to nucleus,
GFP-GUS is excluded from nucleus
unless a nuclear loc. signal is added, then it is only in the nucleus
-uses tobacco etch virus for delivery
-images guard cells in epidermal strips and trichomes
-can use magnetic bead to make cDNA libraries from sorted nuclei for
differential display
-cell type specific promoters (KAT1 in guard cells, commulina yellow mottle
virus promoter is phloem specific)
-will look at nuclear targeting and targeting to nucleus during cell division

PAUL FERRIGNO (Harvard)
-in yeast cdc23-GFP localizes in nucleus
-GFP-cyclin
-also looking at GFP fusions in mutant backgrounds
- do not use adenine mutant strains of yeast as they have increased
background fluorescence.  Also, sick and dying yeast turn yellow.

GREGG GUNDERSEN (Columbia U, New York)
- in mammalian cells, uses GFP-integrin (alpha1 integrin Signal sequence -
GFP- beta1Integrin TMD and cytoplasmic domain)
-in NON-crawling cells adhesion plaques move toward the center of the cell
(0.1-0.3 micron/min.) showed 90 min. time lapse movies
-in crawling cells plaques do not move relative to the substrate, but do
form close to front edge
-microinjected rhodamine-actin and saw that plaques were connected to f-actin
-movie, plaques move on actin, sometimes a plaque on each end
-actin does not appear "behind" the plaques as they move
-interference reflection microscopy showed good contrast Co-loc to
GFP-integrin during slow movement, but when plaques had burst of movement
the contrast was lost, so ECM contact is modulated
-will look at drug effects
-might look at trafficking
-also had movies of intermediate filaments labeled with GFP before and
after antibody injection

JENNIFER LIPPINCOTT-SCHWARTZ (NICHD, NIH)
-see Sept. 4 Nature
-superb movies of VSVG-GFP in ER to GOLGI transport
-used temp. sensitive variant of VSVG: 32 degrees-secr. pathway to cell
surface, 40 degrees- trapped in ER, 15 degrees-in tubular-vesicular body
(co-loc with beta-cop antibody)
-15 then shift to 32, can see movement into golgi region in a stop-n-go
fashion which requires MTs (1.4 micron/sec)
-40 then shift to 32, pre-golgi vesicles pop out of ER and jump onto MTs
and track to golgi 
-also photo-bleaching of golgi and see recovery
- Overexpression of the p50 Subunit of dynactin that builds the motor
together with dynein, thereby abolishing motor function. EFFECT: NO ER to
GOLGI Movement but pre GOLGI "vesicles" build

GEORGE BANTING (U of Bristol)
-TGN38-GFP
-BFA sensitivity
-photobleach  1/4 of TGN, did not see recovery
-showed movies, also see his web site which will soon have movies used
metallothionein promoter to control the amount of produced protein

TED KAHN (The Scripps Research Institute)
-fused GFP to movement protein from TMV watched pattern of loc. throughout
infection
-mutated MP and saw 6 different categories of MP localization
-trying to make dominant negative MP transgenic tobacco for a virus
resistant plant

DALE LAIRD (U of Western Ontario)
-studies gap junctions with GFP-connexin (Cx43)
-GFP-Cx43 is not dominant neg, but still determining if it is functional
(microinjected neurobiotin)
-delete cytoplasmic carboxy terminus and GFP-Cx gets stuck in ER 
        
ROLAND STAUBER (NCI-FCRDC)
-incorporated GFP-Vpr during HIV assembly
-image HIV infection (glowing virions) co-image with marked endosomes
-ConA induced endocytosis can not complement for receptor binding.
-HIV does not colocalize with transferrin receptor after internalization,
which shows that HIV does not get into endosomes (looks like virus enters
cell via membrane fusion)
-HIV-Rev-GFP in cytoplasm and HIV-Rev-BFP in nucleus only, this is live
assay for nuclear entry, when GFP-Rev has nls deleted it still gets into
nucleus but is not retained there, unless Rev-BFP holds Rev-GFP in the
nucleus
-signal is for nuclear retention, not nuclear import

RICHARD WHITE (Mayo Foundation)
-centrin-GFP, has stable cell lines (centrin is a
centriole/centrosome-protein which is upregulated in most breast cancers)
-will see if other cancers have centrosome structure like breast cancer,
which sometimes show several centrioles

DAVID PISTON (Vanderbilt U.)
- general comments on quantitative fluorescence imaging. main points: 
saturation effects (mainly with confocal) and background subtraction.
-GFP-Quantification, comparing wt and EGFP in spectral
properties and minimal amount of detectable molecules
-biophysical characterization of various GFP mutants done under
more standardized conditions: extinction coefficients, bleaching rates,
folding rates, etc.
- optimized filter sets for confocal microscopy of GFP
- tips for quantitation of GFP in cells
-TATA binding protein-GFP function in yeast knockouts
        
JIM HASELOFF (MRC lab, Cambridge) 
-made plant optimized GFPs (remove cryptic intron)
-note that EGFP codon changes also removes cryptic intron which may be
spliced at a low rate in other organisms
-for happy plants GFP in the ER is better than in the cytoplasm, because it
gets into the nucleus
-made enhancer trap lines: GAL4:KanR:UAS driving GFP
-endogenous enhancer drives expression of GAL4 which starts up GFP
expression, screened 7,500 lines and kept 250 (available through stock
center sometime in the next year)
-has GFP expressed in specific cell types (interested in root development)
-can add other genes under UAS control (like diptheria toxin to kill)
-has different lines with GFP in endodermis cortex, pericycle, vascular
bundle, c2 cells and also other lines
-uses tight band pass filter and lines in the argon laser to co-image
mGFP5, mYFP, and mCYP at 477,514 and 458 respectively
-transcription factor (KNAT3-mYFP) is excluded from nucleus in young cells,
but is in nucleus in older cells (has movies of this)
-check out his web site http://brindabella.mrc-lmb.cam.ac.uk/

PETRA SCHWILLE (Cornell U.)
-used fluorescence correlation spectroscopy (FCS) to see blinking GFP
-tau=150 microseconds blinking
-intersystem crossing is ruled out
-most likely explanation is protonation/deprotonation (agrees with pH
sensitivity)

WEDNESDAY

BRETT KING (Stanford) (in Brett's own words)
--using fluorescence upconversion spectroscopy to measure the time-resolved 
emission from GFP, it is shown that for excitation at 398 nm, there is a rapid
decay of emission at 460 nm along with a corresponding rise of emission at
508 nm.  The kinetics of the rise at 508 nm match those of the decay at 460 nm,
indicating that the initially excited state rapidly converts to another 
species.  The excited state conversion rate can be markedly slowed by 
deuterating exchangeable protons, indicating that the conversion involves 
a proton transfer reaction.  We designate the higher energy state A and the 
lower energy state B.  
--this observation along with the slow photoconversion of state A to state B
leads to a model in which the two visible absorption bands correspond to the 
chromophore in two ground state conformations which differ in protonation 
state.
--in order to reconcile the slow photoconversion with the fast conversion in 
the excited state, it is necessary to invoke an intermediate state, I, where
I is an unrelaxed form of B.  The state I evolves to state B upon 
protein relaxation/reorganization around the chromophore.
--therefore, the protein environment preferentially stabilizes one form of the 
chromophore or the other.      
--time-resolved fluorescence experiments on Y66H/Y145F gave no indication of
excited state dynamics, in agreement with predictions based on our model.
--stark spectrocopy was used to measure the magnitude of the change in dipole
moment between the ground and excited states of the lower energy absorption
band B in GFP and of two mutants, S65T and Y66H/Y145F.  
--wt and S65T show similar, substantial Stark effect (7 D) while that for
Y66H/Y145F is smaller (2.5 D).  Using Y66H/Y145F as a model for state A of GFP,
the Stark effect data shows that the charge distribution for states A and B of 
GFP are very different, which helps to rationalize why the protein environment
around the protonated form of the chromophore (state A) is different than 
around the deprotonated form (state B).  
        
H. NIWA (U. of Electro-Commun., Chofu, Tokyo, Japan)
-analyses spectral properties of two model substrates thereby determining the
real structure of the chromophore
-expressed GFP, purified fluorophore containing peptide, and examined 
fluorophore structure outside of the protein molecule
- discrepancy between mass spec and predicted weight indicates peptide has
Cys bonds.
-isolated fluorophore has weak but detectable fluorescence (in aqueous solution)
-spectrum of fluorophore affected by solvent conditions : nonpolar shifts
it to the red.
-Na2S2O4 reversibly reduces the model compound for the chromophore 
with similar spectral effect as in GFP
-showed Japanese news paper article of the GFP glowing mice (not his group)

GOTTFRIED PALM (NCI-FCRDC)
-showed 3D structure of the chromophore with its environment including
H-Acceptors and H-Bonds for S65C and Y66H
-explained difference in spectral properties by different H-bonds
-structures support anionic chromophore for 400 nm absorbing GFP, 
neutral chromophore for 470 nm absorbing GFP. E222 is charged 
complimentarily (anionic for 400 nm, neutral for 470 nm)
-phenol group in wt GFP is replaced by imidazolyl plus water in 
F64L/Y66H structure with otherwise little changes (not so in Y66H/Y145F by 
R. Wachter)

MICHAEL ELOWITZ (Princeton)
-photoactivated GFP with (which blue or UV) filter emission around 600nm
and saw red glowing bacteria (wt/mut1,2,3/S65T/I167T/GFP-uv/cycle 3 were
excited with blue,
wt/GFP-uv/cycle 3 better excitable with UV)
-requires low oxygen environment
-photoactivated GFP forms slowly (tau = 0.7 s) after short irradiation (30 ms)
-GFP is photobleached in its standard emission range 
-did photoactivation/photobleaching of one end of bacterial cell, saw
diffusion of green into bleached area and diffusion of red away from
photoactivated area (rate was about 7 micron squared/sec.)
-published in Current Biology 1997, 7(10)809

BILL WARD (Rutgers)
-showed possibility of dimer formation via hydrophilic and hydrophobic
interfaces
and explained difference in spectral properties of the dimer by pulling the
chromophore via Glu 222 which is connected to 2 residues of the hydrophobic
interface(L221 and F223)
-dimer formation can be followed by gel filtration and change in absorption
peak ratio (470 nm/400 nm)
-hypothesis: in the dimer the interaction of L221 and F223 with its
counterparts from the other subunit pulls E222. This disrupts the
carboxylate - S65 hydrogen bond
-his 3 year old daughter predicted the proper dimer in a drawing (she is 5 now)

JOHN WAMPLER (U of Georgia)
-presented crystal structure with GFP dimers (His taggged GFP, His tag is
not visible in the structure)
-2.7 angstrom resolution
-spectra of GFP in solution and in the crystal are very similar
-the spectra of GFP crystals show no orientation dependency, maybe 
due to the averaging effect of the 12 different molecule orientations (in 
P6(1)22)

EXTRA NOTE:
Hugh Fisher (Ward lab, Rutgers)
-presented a new mutant (F64C) that is brighter
-He was characterizing this the week of the meeting. Maybe he can be
encouraged to post his abstract. (kawasaki at rci.rutgers.edu)

THIS SUMMARY CONTRIBUTED TO BY:
Alvar Carlson acarlson at uoguelph.ca
Neal Gliksman neal_g at image1.com
Daniel Gonzales meton at rci.rutgers.edu
Christine Hellweg elisabet at zedat.fu-berlin.de
Audrey Ichida ichida at biodec.wustl.edu
Eugene Kandel u09577 at uic.edu
Brett King boink at chem.Stanford.EDU
Beat Ludin ludin at fmi.ch
Atsushi Miyawaki me7am at sdcc12.ucsd.edu
Gottfried Palm palm at crysv1.ncifcrf.gov
Winfried Wunderlich wunderlich at nt.imp.univie.ac.at

Good GFP web site (with links) 
http://www.image1.com/products/metagfp/gfplinks.html
(This is not an endorsement and Neal had nothing to do with this
recommendation. Consider this "word of mouth".)

Please direct any complaints to ichida at biodec.wustl.edu





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