Characterization of Enzyme Motions by Solution NMR Relaxation Dispersion

Abstract

In many enzymes, conformational changes that occur along the reaction coordinate can pose a bottleneck to the rate of conversion of substrates to products. Characterization of these rate-limiting protein motions is essential for obtaining a full understanding of enzyme-catalyzed reactions. Solution NMR experiments such as the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo or off-resonance R 1rho pulse sequences enable quantitation of protein motions in the time range of microseconds to milliseconds. These experiments allow characterization of the conformational exchange rate constant, k ex, the equilibrium populations of the relevant conformations, and the chemical shift differences (Deltaomega) between the conformations. The CPMG experiments were applied to the backbone N-H positions of ribonuclease A (RNase A). To probe the role of dynamic processes in the catalytic cycle of RNase A, stable mimics of the apo enzyme (E), enzyme-substrate (ES) complex, and enzyme-product (EP) complex were formed. The results indicate that the ligand has relatively little influence on the kinetics of motion, which occurs at 1700 s (-1) and is the same as both k cat, and the product dissociation rate constant. Instead, the effect of ligand is to stabilize one of the pre-existing conformations. Thus, these NMR experiments indicate that the conformational change in RNase A is ligand-stabilized and does not appear to be ligand-induced. Further evidence for the coupling of motion and enzyme function comes from the similar solvent deuterium kinetic isotope effect on k ex derived from the NMR measurements and k cat from enzyme kinetic studies. This isotope effect of approximately 2 depends linearly on solvent deuterium content suggesting the involvement of a single proton in RNase A motion and function. Moreover, mutation of His48 to alanine eliminates motion in RNase A and decreases the catalytic turnover rate indicating the involvement of His48, which is far from the active site, in coupling motion and function. For the enzyme triosephosphate isomerase (TIM), the opening and closing motion of a highly conserved active site loop (loop 6) has been implicated in many studies to play an important role in the catalytic cycle of the enzyme. Off-resonance R 1rho experiments were performed on TIM, and results were obtained for amino acid residues in the N-terminal (Val167), and C-terminal (Lys174, Thr177) portions of loop 6. The results indicate that all three loop residues move between the open and closed conformation at about 10,000 s (-1), which is the same as the catalytic rate constant. The O (eta) atom of Tyr208 provides a hydrogen bond to stabilize the closed form of loop 6 by interacting with the amide nitrogen of Ala176; these atoms are outside of hydrogen bonding distance in the open form of the enzyme. Mutation of Tyr208 to phenylalanine results in significant loss of catalytic activity but does not appear to alter the kex value of the N-terminal part of loop 6. Instead, removal of this hydrogen bond appears to result in an increase in ...

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

“…In contrast, the apo or GDP-bound forms show greater flexibility at the Nterminal β1-strand and more pronounced fluctuations in dynamics throughout the protein. To probe dynamics in the millisecond-tomicrosecond time scale, we performed 15 N and 13 C CPMG relaxation dispersion experiments, which are particularly suitable for probing conformational changes such as protein-folding events (32) and motions associated with enzyme catalysis (33). Initially, we recorded 15 N CPMG relaxation dispersion experiments on the apo, GDP-, and GTPγS-bound forms on backbone amide resonances in the protein.…”

Structure of the Bacillus anthracis Sortase A Enzyme Bound to Its Sorting Signal

“…3C). Because the Model-free analysis only indirectly reveals the presence of R ex contributions to relaxation, we also performed constantrelaxation time Carr-Purcell-Meiboom-Gill (CPMG) experiments (64) that probe motions occurring on time scales ranging from 0.1 to 10 ms (63,65,66). However, analysis of 15 N-CPMG relaxation dispersion curves did not reveal the presence of R ex in the N-terminal appendage (data not shown).…”

Structure and Dynamics of Proteins from Nuclear Magnetic Resonance Spectroscopy

“…-is a technique used to obtain kinetic, thermodynamic, and structural information and applies to exchange processes occurring in the 0.3 to 10 millisecond time frame (Kleckner & Foster, 2011;Loria et al, 2008). The purpose of this particular experiment is to use a series of spin echo pulses to transverse magnetization during a relaxation delay in order to refocus exchange broadening (Kleckner & Foster, 2011).…”

“…-may be used to study exchange processes that occur within the 20 to 100 microsecond range (Kleckner & Foster, 2011;Loria et al, 2008). This particular experiment is very similar to that of CPMG, the only difference is in the range of the spin echo pulses used (25-1200 Hz for CPMG and 1-50kHz for RFRD), this allows RF RD to study exchange events via the same principles as CPMG, but on a faster timescale (Kleckner & Foster, 2011).…”