On the Use of Side-Chain NMR Relaxation Data to Derive Structural and Dynamical Information on Proteins: A Case Study...

Values of $$ and $$ order parameters derived from NMR relaxation measurements on proteins cannot be used straightforwardly to determine protein structure because they cannot be related to a single protein structure, but are defined in terms of an average over a conformational ensemble. Molecular dynamics simulation can generate a conformational ensemble and thus can be used to restrain $$ and $$ order parameters towards experimentally derived target values $$ (exp) and $$ (exp). Application of $$ and $$ order-parameter restraining MD simulation to bond vectors in 63 side chains of the protein hen egg white lysozyme using 51 $$ (exp) target values and 28 $$ (exp) target values shows that a conformational ensemble compatible with the experimentally derived data can be obtained by using this technique. It is observed that $$ order-parameter restraining of C-H bonds in methyl groups is less reliable than $$ order-parameter restraining because of the possibly less valid assumptions and approximations used to derive experimental $$ (exp) values from NMR relaxation measurements and the necessity to adopt the assumption of uniform rotational motion of methyl C-H bonds around their symmetry axis and of the independence of these motions from each other. The restrained simulations demonstrate that side chains on the protein surface are highly dynamic. Any hydrogen bonds they form and that appear in any of four different crystal structures, are fluctuating with short lifetimes in solution.