XXXX bond isomerization in basic pancreatic trypsin inhibitor: multisite chemical exchange quantified by CPMG relaxation dispersion and chemical shift modeling.

@entity2569 @entity534 @entity1520 @entity13140
Conformational changes occurring on the microsecond-millisecond time scale in basic pancreatic trypsin inhibitor (BPTI) are investigated using nuclear magnetic resonance spectroscopy. The rczz CPMG experiment (Wang, C.; Grey, M. J.; Palmer, A. G. J. Biomol. NMR 2001, 21, 361-366) is used to record @entity13140 spin relaxation dispersion data, R(ex)(1/tau(cp)), in which 1/tau(cp) is the pulsing rate in the CPMG sequence, at two static magnetic fields, 11.7 and 14.1 T, and three temperatures, 280, 290, and 300 K. These data are used to characterize the kinetics and mechanism of chemical exchange line broadening of the backbone @entity13140 spins of @entity2569 14, Lys 15, @entity2569 38, and @entity534 39 in BPTI. Line broadening is found to result from two processes: the previously identified isomerization of the @entity2569 38 side chain between chi(1) rotamers (Otting, G.; Liepinsh, E.; W  thrich, K. Biochemistry 1993, 32, 3571-3582) and a previously uncharacterized process on a faster time scale. At 300 K, both processes contribute significantly to the relaxation dispersion for @entity2569 14 and an analytical expression for a linear three-site exchange model is used to analyze the data. At 280 K, isomerization of the @entity2569 38 side chain is negligibly slow and the faster process dominates the relaxation dispersion for all four spins. Global analysis of the temperature and static field dependence of R(ex)(1/tau(cp)) for @entity2569 14 and Lys 15 is used to determine the activation parameters and chemical shift changes for the previously uncharacterized chemical exchange process. Through an analysis of a database of chemical shifts, @entity13140 chemical shift changes for @entity2569 14 and Lys 15 are interpreted to result from a chi(1) rotamer transition of @entity2569 14 that converts the @entity2569 14- @entity2569 38 @entity1520 bond between right- and left-handed conformations. At 290 K, isomerization of @entity2569 14 occurs with a forward and reverse rate constant of 35 s(-1) and 2500 s(-1), respectively, a time scale more than 30-fold faster than the @entity2569 38 chi(1) isomerization. A comparison of the kinetics and thermodynamics for the transitions between the two alternative @entity2569 14- @entity2569 38 conformations highlights the factors that affect the contribution of @entity1520 bonds to protein stability.
