Methyl Rotation Barriers in Proteins from ²H Relaxation Data. Implications for Protein Structure

Abstract

Side-chain 2H and backbone 15N relaxation data have been collected at multiple temperatures in the samples of the SH3 domain from alpha-spectrin. Combined analyses of the data allowed for determination of the temperature-dependent correlation times tauf characterizing fast methyl motion. Molecular dynamics simulations confirmed that tauf are dominated by methyl rotation; the corresponding activation energies approximate methyl rotation barriers. For 33 methyl groups in the alpha-spectrin SH3 domain the average barrier height was thus determined to be 2.8 +/- 0.9 kcal/mol. This value is deemed representative of the "fluid" hydrophobic protein core where some barriers are increased and others are lowered because of the contacts with surrounding atoms, but there is no local order that could produce systematically higher (lower) barriers. For comparison, the MD simulation predicts the average barrier of 3.1 kcal/mol (calculated via the potential of mean force) or 3.4-3.5 kcal/mol (rigid barriers after appropriate averaging over multiple MD snapshots). The latter result prompted us to investigate rigid methyl rotation barriers in a series of NMR structures from the Protein Databank. In most cases the barriers proved to be higher than expected, 4-6 kcal/mol. To a certain degree, this is caused by tight packing of the side chains in the NMR structures and stems from the structure calculation procedure where the coordinates are first annealed toward the temperature of 0 K and then subjected to energy minimization. In several cases the barriers >10 kcal/mol are indicative of van der Waals violations. The notable exceptions are (i) the structures solved using the GROMOS force field where tight methyl packing is avoided (3.0-3.6 kcal/mol) and (ii) the structure solved by means of the dynamic ensemble refinement method (Lindorff-Larsen, K.; Best, R. B.; DePristo, M. A.; Dobson, C. M.; Vendruscolo, M. Nature 2005, 433, 128) (3.5 kcal/mol). These results demonstrate that methyl rotation barriers, derived from the experiments that are traditionally associated with studies of protein dynamics, can be also used in the context of structural work. This is particularly interesting in view of the recent efforts to incorporate dynamics data in the process of protein structure elucidation.

Toward Quantitative Interpretation of Methyl Side-Chain Dynamics from NMR by Molecular Dynamics Simulations

“…15,22 The rmsd of 0.06 between experimental and simulated J(0) values is smaller than the rmsd between the experimental J(0) of the Ca 2+ -bound and the apo state. The largest discrepancy is found for Thr45 (boxed data point) whose time correlation function is not wellconverged; 3 that is, its S 2 value has a low precision (see Supporting Information).…”

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

“…75 Note that the correlation for Cys 3 decays noticeably faster than that for Ile 53 (effective decay time 6.8 ps vs. 10.7 ps). Indeed, for two nearby particles mutual diffusion leads to rapid modulation of the dipolar interaction; conversely, for two distant particles, the modulation is slow.…”

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

“…Surprisingly, however, the 2 H NMR relaxation studies show that there are site-specific variations in the dynamics, as manifested by both the pre-exponential factor and activation barriers for the retinal motions (Fig 6). The site-specific variations in the methyl barriers in a protein have been seen also in the SH3 domain from α-spectrin [167]. Activation energies correspond to the temperature dependence of the relaxation rates and derived correlation times and assume an Arrhenius activation law (vide supra).…”