Motions of proteins are essential for the performance of their functions. Aliphatic protein side chains and their motions play critical roles in protein interactions: for recognition and binding of partner molecules at the surface or serving as an entropy reservoir within the hydrophobic core. Here, we present a new NMR method based on high-resolution relaxometry and high-field relaxation to determine quantitatively both motional amplitudes and time scales of methyl-bearing side chains in the picosecond-to-nanosecond range. We detect a wide variety of motions in isoleucine side chains in the protein ubiquitin. We unambiguously identify slow motions in the low nanosecond range, which, in conjunction with molecular dynamics computer simulations, could be assigned to transitions between rotamers. Our approach provides unmatched detailed insight into the motions of aliphatic side chains in proteins and provides a better understanding of the nature and functional role of protein side-chain motions.

Functional protein motions covering a wide range of time scales can be studied, among other techniques, by NMR and by molecular dynamics (MD) computer simulations. MD simulations of proteins now routinely extend into the hundreds of nanoseconds time scale range exceeding the overall tumbling correlation times of proteins in solution by several orders of magnitude. This provides a unique opportunity to rigorously validate these simulations by quantitative comparison with model-free order parameters derived from NMR relaxation experiments. However, presently there is no consensus on how such a comparison is best done. We address here how this can be accomplished in a way that is both efficient and objective. For this purpose, we analyze (15)N R1 and R2 and heteronuclear {(1)H}-(15)N NOE NMR relaxation parameters computed from 500 ns MD trajectories of 10 different protein systems using the model-free analysis. The resulting model-free S(2) order parameters are then used as targets for S(2) values computed directly from the trajectories by the iRED method by either averaging over blocks of variable lengths or by using exponentially weighted snapshots (wiRED). We find that the iRED results are capable of reproducing the target S(2) values with high accuracy provided that the averaging window is chosen 5 times the length of the overall tumbling correlation time. These results provide useful guidelines for the derivation of NMR order parameters from MD for a meaningful comparison with their experimental counterparts.

The flexible nature of protein loops and the time scales of their dynamics are critical for many biologically important events at the molecular level, such as protein interaction and recognition processes. In order to obtain a predictive understanding of the dynamic properties of loops, 500 ns molecular dynamics (MD) computer simulations of 38 different proteins were performed and validated using NMR chemical shifts. A total of 169 loops were analyzed and classified into three types, namely fast loops with correlation times <10 ns, slow loops with correlation times between 10 and 500 ns, and loops that are static over the course of the whole trajectory. Chemical and biophysical loop descriptors, such as amino-acid sequence, average 3D structure, charge distribution, hydrophobicity, and local contacts were used to develop and parametrize the ToeLoop algorithm for the prediction of the flexibility and motional time scale of every protein loop, which is also implemented as a public Web server (http://spin.ccic.ohio-state.edu/index.php/loop). The results demonstrate that loop dynamics with their time scales can be predicted rapidly with reasonable accuracy, which will allow the screening of average protein structures to help better understand the various roles loops can play in the context of protein-protein interactions and binding.

Functional motions of 15N-labeled proteins can be monitored by solution NMR spin relaxation experiments over a broad range of timescales. These experiments however typically take of the order of several days to a week per protein. Recently, NMR chemical exchange saturation transfer (CEST) experiments have emerged to probe slow millisecond motions complementing R1ρ and CPMG-type experiments. CEST also simultaneously reports on site-specific R1 and R2 parameters. It is shown here how CEST-derived R1, R2 relaxation parameters can be measured within few hours at an accuracy comparable to traditional relaxation experiments. Using a “lean” version of the model-free approach S2 order parameters can be determined that match those from the standard model-free approach applied to 15N R1, R2, and {1H}-15N NOE data. The new methodology, which is demonstrated for ubiquitin and arginine kinase (42 kDa), should serve as an effective screening tool of protein dynamics from ps to ms timescales.

Supplementary data are available at Bioinformatics online.