Some problems with RET data interpretation

Kyle Legate legatek at mcmail.cis.mcmaster.ca
Thu Jan 23 14:13:57 EST 2003


I have been asked to crosspost my question to the fluorescence and methods
newsgroups, so apologies to those receiving this message twice. Hopefully
it will generate twice the help!
	I am performing resonance energy transfer (RET) experiments on my
protein to examine binding kinetics to fluorescently-labelled nucleotide
(mant-GTP). My protein contains a single tryptophan, which should make
analysis simple, but I am running into some difficulties.
	I excite my sample at 280nm to excite the trp, which emits around
330nm to excite the mant, which emits around 440nm. I am analysing
mant-GTP binding by measurement of the increase in mant fluorescence over
time. I observe a concomitant decrease in trp emission due to quenching by
the mant, as I expect. However, I also observe a time-dependent decrease
in trp fluorescence that occurs in the absence of acceptor fluorophore.
This results in poor correlation between loss of donor emission and gain
of acceptor emission (for example, while I notice an initial increase in
mant-GTP binding followed by a gradual loss of binding (dying protein?),
the decrease in trp emission continues throughout the assay).
	I thought this might be due to gradual misfolding of the protein
in
my assay conditions so to test for this I incubated protein alone in the
presence of 2M guanidine HCl. What I notice is an immediate red shift in
the emission spectrum, consistent with what one would expect from an
increase in solvent contact (ie. the denaturation is working) and a
continued time dependent decrease of trp fluorescence, though a more
modest decrease than that observed in normal assay conditions. I do not
notice a red shift in the absence of guanidine, so the misfolding
hypothesis is questionable.
	Does anyone have an explanation for what I am seeing re: decreased
trp fluorescence? Could it be an indication of protein aggregation and
subsequent
quenching of trp? I am closing the laser gate between readings to minimise
the effect of photobleaching. I could post data examples on the web and
provide a pointer here if anyone thinks it will help.

... . . .  .  .  .    .    .    .     .     .     .      .      .      .
legatek at mcmaster.ca		Kyle Legate            legatek at hotmail.com

   Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
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