Solid-state NMR approaches to internal dynamics of proteins: from picoseconds to microseconds and seconds

Solid-state nuclear magnetic resonance (NMR) spectroscopy has matured to the point that it is possible to determine the structure of proteins in immobilized states, such as within microcrystals or embedded in membranes. Currently, researchers continue to develop and apply NMR techniques that can deliver site-resolved dynamic information toward the goal of understanding protein function at the atomic scale. As a widely-used, natural approach, researchers have mostly measured longitudinal (T1) relaxation times, which, like in solution-state NMR, are sensitive to picosecond and nanosecond motions, and motionally averaged dipolar couplings, which provide an integral amplitude of all motions with a correlation time of up to a few microseconds. While overall Brownian tumbling in solution mostly precludes access to slower internal dynamics, dedicated solid-state NMR approaches are now emerging as powerful new options. In this Account, we give an overview of the classes of solid-state NMR experiments that have expanded the accessible range correlation times from microseconds to many milliseconds. The measurement of relaxation times in the rotating frame, T1ρ, now allows researchers to access the microsecond range. Using our recent theoretical work, researchers can now quantitatively analyze this data to distinguish relaxation due to chemical-shift anisotropy (CSA) from that due to dipole-dipole couplings. Off-resonance irradiation allows researchers to extend the frequency range of such experiments. We have built multidimensional analogues of T2-type or line shape experiments using variants of the dipolar-chemical shift correlation (DIPSHIFT) experiment that are particularly suited to extract intermediate time scale motions in the millisecond range. In addition, we have continuously improved variants of exchange experiments, mostly relying on the recoupling of anisotropic interactions to address ultraslow motions in the ms to s ranges. The NH dipolar coupling offers a useful probe of local dynamics, especially with proton-depleted samples that suppress the adverse effect of strong proton dipolar couplings. We demonstrate how these techniques have provided a concise picture of the internal dynamics in a popular model system, the SH3 domain of α-spectrin. T1-based methods have shown that large-amplitude bond orientation fluctuations in the picosecond range and slower 10 ns low-amplitude motions coexist in these structures. When we include T1ρ data, we observe that many residues undergo low amplitude motions slower than 100 ns. On the millisecond to second scale, mostly localized but potentially cooperative motions occur. Comparing different exchange experiments, we found that terminal NH2 groups in side chains can even undergo a combination of ultraslow large-angle two-site jumps accompanied by small-angle fluctuations that occur 10 times more quickly.