Disulfide Bond Isomerization in Basic Pancreatic Trypsin Inhibitor: Multisite Chemical Exchange Quantified by CPMG Relaxation Dispersion and Chemical Shift Modeling
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- 作者：Michael J. Grey ; Chunyu Wang ; and Arthur G. Palmer ; III
- 刊名：Journal of the American Chemical Society
- 出版年：2003
- 出版时间：November 26, 2003
- 年：2003
- 卷：125
- 期：47
- 页码：14324 - 14335
- 全文大小：277K
- 年卷期：v.125,no.47(November 26, 2003)
- ISSN：1520-5126
文摘
Conformational changes occurring on the microsecond-millisecond time scale in basic pancreatictrypsin inhibitor (BPTI) are investigated using nuclear magnetic resonance spectroscopy. The rczz CPMGexperiment (Wang, C.; Grey, M. J.; Palmer, A. G. J. Biomol. NMR 2001, 21, 361-366) is used to record15N spin relaxation dispersion data, Rex(1/
cp), in which 1/cp is the pulsing rate in the CPMG sequence, attwo static magnetic fields, 11.7 and 14.1 T, and three temperatures, 280, 290, and 300 K. These data areused to characterize the kinetics and mechanism of chemical exchange line broadening of the backbone15N spins of Cys 14, Lys 15, Cys 38, and Arg 39 in BPTI. Line broadening is found to result from twoprocesses: the previously identified isomerization of the Cys 38 side chain between 
1 rotamers (Otting,G.; Liepinsh, E.; Wüthrich, K. Biochemistry 1993, 32, 3571-3582) and a previously uncharacterized processon a faster time scale. At 300 K, both processes contribute significantly to the relaxation dispersion for Cys14 and an analytical expression for a linear three-site exchange model is used to analyze the data. At 280K, isomerization of the Cys 38 side chain is negligibly slow and the faster process dominates the relaxationdispersion for all four spins. Global analysis of the temperature and static field dependence of Rex(1/cp) forCys 14 and Lys 15 is used to determine the activation parameters and chemical shift changes for thepreviously uncharacterized chemical exchange process. Through an analysis of a database of chemicalshifts, 15N chemical shift changes for Cys 14 and Lys 15 are interpreted to result from a 1 rotamer transitionof Cys 14 that converts the Cys 14-Cys 38 disulfide bond between right- and left-handed conformations.At 290 K, isomerization of Cys 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 Cys 38 1 isomerization. A comparison of thekinetics and thermodynamics for the transitions between the two alternative Cys 14-Cys 38 conformationshighlights the factors that affect the contribution of disulfide bonds to protein stability.
cp), in which 1/cp is the pulsing rate in the CPMG sequence, attwo static magnetic fields, 11.7 and 14.1 T, and three temperatures, 280, 290, and 300 K. These data areused to characterize the kinetics and mechanism of chemical exchange line broadening of the backbone15N spins of Cys 14, Lys 15, Cys 38, and Arg 39 in BPTI. Line broadening is found to result from twoprocesses: the previously identified isomerization of the Cys 38 side chain between 1 rotamers (Otting,G.; Liepinsh, E.; Wüthrich, K. Biochemistry 1993, 32, 3571-3582) and a previously uncharacterized processon a faster time scale. At 300 K, both processes contribute significantly to the relaxation dispersion for Cys14 and an analytical expression for a linear three-site exchange model is used to analyze the data. At 280K, isomerization of the Cys 38 side chain is negligibly slow and the faster process dominates the relaxationdispersion for all four spins. Global analysis of the temperature and static field dependence of Rex(1/cp) forCys 14 and Lys 15 is used to determine the activation parameters and chemical shift changes for thepreviously uncharacterized chemical exchange process. Through an analysis of a database of chemicalshifts, 15N chemical shift changes for Cys 14 and Lys 15 are interpreted to result from a 1 rotamer transitionof Cys 14 that converts the Cys 14-Cys 38 disulfide bond between right- and left-handed conformations.At 290 K, isomerization of Cys 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 Cys 38 1 isomerization. A comparison of thekinetics and thermodynamics for the transitions between the two alternative Cys 14-Cys 38 conformationshighlights the factors that affect the contribution of disulfide bonds to protein stability.