Tryptophan rotamers that report the conformational dynamics of proteins*
Judit Fidy1,**, Monique Laberge1, Beata Ullrich1,
Laszlo Polgar2, Zoltan Szeltner2, Jacques Gallay3,
and Michel Vincent3
1Institute of Biophysics
and Radiation Biology, Faculty of General Medicine, Semmelweis University,
Budapest, H-1444, P.O.B. 263, Hungary; 2Institute of Enzymology,
Biological Research Center, HAS, Budapest, H-1518, P.O.B. 7, Hungary;
3LURE, Université Paris-Sud, Bâtiment 209D,
Orsay, F-91405, France
Abstract: The binding of acetylpepstatin to the Q7K/L33I/L63I
mutant of HIV-1 protease was studied by fluorescence, phosphorescence,
and 500-ps molecular dynamics. The protease is a homodimer with two
tryptophans per monomer. Maximum entropy method (MEM) analysis and acrylamide
quenching results show two tryptophyl, tryptophan (Trp) populations
in the apoenzyme that merge into one in the complex. These results are
in agreement with molecular dynamics simulations indicative of Trp asymmetry
in the apoenzyme as revealed by the occurrence of nonequivalent Trp42
indole rotamer interconversions, not observed for the complex. Analysis
of the local Trp42B environments of the apoenzyme with respect to possible
quencher groups shows that the c2 interconversions do not
influence the lifetime, while the c1 interconversions do.
Upon binding the inhibitor, Trp42B acquires a single conformation with
the same lifetime and orientation as that of Trp42, and also with less
quenching accessibility. Thus, protein conformational dynamics become
constrained with inhibitor binding. This conclusion is supported by
red-edge effect experiments and phosphorescence lifetime measurements.
The low temperature tp (~5.8 s) is quenched to ~200 ms as
protein motions become activated around the glass transition temperature.
In the case of the complex, the phosphorescence lifetime data show a
more cooperative activation of the quenching mechanisms.
*Lecture presented at the XVIIIth IUPAC Symposium on
Photochemistry, Dresden, German , 22-27 July 2000.Other presentations
are published in this issue, pp.395-548.
** Corresponding author