GFP

AMI ichida at BIOMAIL.UCSD.EDU
Tue Nov 4 15:20:17 EST 1997


Hugh Fisher has forwarded his abstract from the GFP meeting to me. It
should be included in the summary posted earlier:

   A single round of DNA shuffling (Stemmer, Nature 370; 389-390) applied
to
the Y66H mutant gave rise to a new type of chromophore mutant (NSS#1). A
single colony displaying unusually rapid fluorescence development and a
greater than usual degree of thermotolerance was isolated. Spectral
analysis
could not distinguish between the new mutant and wt GFP.  The improvement
in
fluorescence yield was independent of the promoter used. Dynamic
whole-cell
assays at 37!C showed a several-fold increase in fluorescence yield.
Sequencing analysis demonstrated that the phenylalanine at position 64 had
been substituted by a cysteine (F64C). The benefits are also observed in
the
S65T (Heim, et al. Nature 373:663-664.) mutant  (F64C/S65T; aka NSST). 

   Data generated during this symposium have suggested a possible
mechanism
to account for this improvement. NSS#1 apparently has a lower oxygen
requirement for internal catalysis, suggesting that the F64C mutation
assists
somehow in the internal dehydration/ cyclization reaction. If this
hypothesis
is correct, we may be the first to have generated a mutant with an
improved
catalytic mechanism.






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