Characterizing Protein Dynamics with NMR R 1ρ Relaxation Experiments

Abstract

The measurement of R , the longitudinal relaxation rate constant in the rotating frame, is one of the few available methods to characterize the μs-ms functional dynamics of biomolecules. Here, we focus onN R experiments for protein NH groups. We present protocols for both on- and off-resonance N R measurements needed for relaxation dispersion studies, and describe the data analysis for extracting kinetic and thermodynamic parameters characterizing the motional processes.

Two-dimensional NMR lineshape analysis of single, multiple, zero and double quantum correlation experiments

“…a spin that is interconverting between multiple states in dynamic equilibrium) is well known to provide a powerful spectroscopic probe of the underlying exchange process. This lineshape may be modulated by a variety of means, including rf pulse sequences such as CPMG pulse trains (Gopalan et al 2018), R 1 spin locks (Massi and Peng 2018;Xue 2015) or saturation with a single frequency or frequency comb (Vallurupalli et al 2012;Fawzi et al 2011;Yuwen et al 2018); field shuttling (Cousin 2016); or variation of external parameters such as protein or ligand concentration or temperature (Waudby et al 2016(Waudby et al , 2019. The resulting modulations may then be fitted to determine details of the exchange process such as chemical shift differences, populations of states, and the rate of exchange between them.…”

NMR Lineshape Analysis of Intrinsically Disordered Protein Interactions

“…The dependence of the observed NMR spectrum on the kinetics of the exchange process as well as the thermodynamics (the bound population) and structure (chemical shift differences and linewidths) is the essential reason why NMR spectroscopy provides a powerful tool to characterize molecular equilibria and binding reactions. The evolution of magnetization in an exchanging system may also be manipulated with rf fields and pulses, which has given rise to a range of methods such as exchange spectroscopy (EXSY) [22][23][24], CPMG or R 1ρ relaxation dispersion [25][26][27][28][29], and chemical/dark-state exchange saturation transfer (CEST and DEST) [20,30,31], which may be used to characterize dynamic equilibria even within single samples [32]. However, in systems where titrations can be performed, i.e., where the concentration of one or more components can be altered to modulate the equilibrium, NMR titrations using standard 2D correlation experiments provide a powerful approach to the analysis of exchange that is often simpler and more intuitive to analyze than the methods described above.…”

Two-dimensional NMR lineshape analysis of single, multiple, zero and double quantum correlation experiments

“…a spin that is interconverting between multiple states in dynamic equilibrium) is well known to provide a powerful spectroscopic probe of the underlying exchange process. This lineshape may be modulated by a variety of means, including rf pulse sequences such as CPMG pulse trains 1 , R 1ρ spin locks 2,3 or saturation with a single frequency or frequency comb [4][5][6] ; field shuttling 7 ; or variation of external parameters such as protein or ligand concentration or temperature 8,9 . The resulting modulations may then be fitted to determine details of the exchange process such as chemical shift differences, populations of states, and the rate of exchange between them.…”