Methyl rotation barriers in proteins from 2H relaxation data. Implications for protein structure.

Side-chain 2H and backbone 15N relaxation data have been collected at multiple temperatures in the samples of the SH3 domain from α-spectrin. Combined analyses of the data allowed for determination of the temperature-dependent correlation times τf characterizing fast methyl motion. Molecular dynamics simulations confirmed that τf are dominated by methyl rotation; the corresponding activation energies approximate methyl rotation barriers. For 33 methyl groups in the α-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...