Publications

NMR spin relaxation experiments are used to characterize the dynamics of the backbone of ubiquitin. Chemical exchange processes affecting residues Ile 23, Asn 25, Thr 55, and Val 70 are characterized using on- and off-resonance rotating-frame 15N R1rho relaxation experiments to have a kinetic exchange rate constant of 25,000 sec(-1) at 280 K. The exchange process affecting residues 23, 25, and 55 appears to result from disruption of N-cap hydrogen bonds of the alpha-helix and possibly from repacking of the side chain of Ile 23. Chemical exchange processes affecting other residues on the surface of ubiquitin are identified using 1H-15N multiple quantum relaxation experiments. These residues are located near or at the regions known to interact with various enzymes of the ubiquitin-dependent protein degradation pathway.

Transverse relaxation optimized NMR spectroscopy (TROSY) techniques for (1)H-(15)N backbone amide moieties and for (13)CH(3) methyl groups have permitted the development of Hahn spin echo and Carr-Purcell-Meiboom-Gill (CPMG) experiments for characterizing chemical exchange kinetic phenomena on microsecond-millisecond time scales in proteins with molecular masses >50 kDa. This chapter surveys the theoretical bases for TROSY in spin systems subject to chemical exchange linebroadening, the experimental methods that have been developed to quantitatively characterize chemical exchange in large proteins, and the emerging applications to triose phosphate isomerase, hemoglobin, and malate synthase G, with molecular masses ranging from 54 to 82 kDa.

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 (15)N 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 (15)N spins of Cys 14, Lys 15, Cys 38, and Arg 39 in BPTI. Line broadening is found to result from two processes: the previously identified isomerization of the Cys 38 side chain between chi(1) rotamers (Otting, G.; Liepinsh, E.; Wüthrich, 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 Cys 14 and an analytical expression for a linear three-site exchange model is used to analyze the data. At 280 K, isomerization of the Cys 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 Cys 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, (15)N chemical shift changes for Cys 14 and Lys 15 are interpreted to result from a chi(1) rotamer transition of 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 chi(1) isomerization. A comparison of the kinetics and thermodynamics for the transitions between the two alternative Cys 14-Cys 38 conformations highlights the factors that affect the contribution of disulfide bonds to protein stability.

Improved relaxation-compensated Carr-Purcell-Meiboom-Gill pulse sequences are reported for studying chemical exchange of backbone 15N nuclei. In contrast to the original methods [J. P. Loria, M. Rance, and A. G. Palmer, J. Am. Chem. Soc. 121, 2331-2332 (1999)], phenomenological relaxation rate constants obtained using the new sequences do not contain contributions from 1H-1H dipole-dipole interactions. Consequently, detection and quantification of chemical exchange processes are facilitated because the relaxation rate constant in the limit of fast pulsing can be obtained independently from conventional 15N spin relaxation measurements. The advantages of the experiments are demonstrated using basic pancreatic trypsin inhibitor.