Observation of Sub-Microsecond Protein Methyl-Side Chain Dynamics by Nanoparticle-Assisted NMR Spin Relaxation

Abstract

Amino-acid side-chain properties in proteins are key determinants of protein function. NMR spin relaxation of side chains is an important source of information about local protein dynamics and flexibility. However, traditional solution NMR relaxation methods are most sensitive to sub-nanosecond dynamics lacking information on slower ns−μs time-scale motions. Nanoparticle-assisted NMR spin relaxation (NASR) of methyl-side chains is introduced here as a window into these ns−μs dynamics. NASR utilizes the transient and nonspecific interactions between folded proteins and slowly tumbling spherical nanoparticles (NPs), whereby the increase of the relaxation rates reflects motions on time scales from ps all the way to the overall tumbling correlation time of the NPs ranging from hundreds of ns to μs. The observed motional amplitude of each methyl group can then be expressed by a model-free NASR S 2 order parameter. The method is demonstrated for 2H-relaxation of CH2D methyl moieties and cross-correlated relaxation of CH3 groups for proteins Im7 and ubiquitin in the presence of anionic silica-nanoparticles. Both types of relaxation experiments, dominated by either quadrupolar or dipolar interactions, yield highly consistent results. Im7 shows additional dynamics on the intermediate time scales taking place in a functionally important loop, whereas ubiquitin visits the majority of its conformational substates on the sub-ns time scale. These experimental observations are in good agreement with 4–10 μs all-atom molecular dynamics trajectories. NASR probes side-chain dynamics on a much wider range of motional time scales than previously possible, thereby providing new insights into the interplay between protein structure, dynamics, and molecular interactions that govern protein function.

Quantitative Multistate Binding Model of Silica Nanoparticle–Protein Interactions Obtained from Multinuclear Spin Relaxation

“…Notably, the bound rotational correlation time is very close to the theoretical correlation time of 910 ns predicted by the Stokes–Einstein–Debye equation for nanoparticles with a 20 nm diameter in H 2 O used for the experiments. The small bound population (2.04%) is also reasonable as it is consistent with the previously observed invariance of longitudinal relaxation rates in the presence and absence of nanoparticles . Finally, the free-to-encounter rate of approach, k FE = 51.7 s –1 , is well within the diffusion limit of k FE,diff = 1270 s –1 determined according to Alberty and Hammes …”

“…The small bound population (2.04%) is also reasonable as it is consistent with the previously observed invariance of longitudinal relaxation rates in the presence and absence of nanoparticles. 32 Finally, the free-to-encounter rate of approach, k FE = 51.7 s −1 , is well within the diffusion limit of k FE,diff = 1270 s −1 determined according to Alberty and Hammes. 38 In our previous work on the modeling of NASR data using SLE, it was noted that relaxation rates behave similarly for fast and slow exchange rates.…”

“…Additionally, this difference in relaxation also depends on the kinetics of binding when more sophisticated motional models are used for spin relaxation data interpretation. 31 In our recent work, we presented a set of NASR experiments utilizing methyl 13 C− 1 H dipole−dipole cross-correlation rates Γ and methyl 2 H transverse relaxation rates R(D + ) (abbreviated as R + below) for ubiquitin, henceforth referred to as generic relaxation rates R. 32 There we noted a systematic positive offset in the linear correlation of NASR rates versus Lipari−Szabo order parameter S axis 2 . A simple two-site exchange model predicts that NASR relaxation rates follow the relationship 9…”

“…In our recent work, we presented a set of NASR experiments utilizing methyl 13 C– 1 H dipole–dipole cross-correlation rates Γ and methyl 2 H transverse relaxation rates R (D + ) (abbreviated as R + below) for ubiquitin, henceforth referred to as generic relaxation rates R . There we noted a systematic positive offset in the linear correlation of NASR rates versus Lipari–Szabo order parameter S axis 2 .…”

How Much Entropy Is Contained in NMR Relaxation Parameters?

“…As these studies show, viscogens may not be the best choice for the purpose of slowing molecular tumbling. As an alternative to increasing solvent viscosity, Brüschweiler and co-workers recently took a different approach, where they study proteins that exchange between solution and the corona of a nanoparticle. , They set up a system where the protein spends a marginal amount of time (ca. 1%) in the bound state, but this stopover leaves a signature of the internal dynamics of the protein-nanoparticle complex.…”