A REASSESSMENT OF THE STRUCTURE OF CHYMOTRYPSIN INHIBITOR-2 (CI-2) USING TIME-AVERAGED NMR RESTRAINTS

Chymotrypsin inhibitor 2 (CI-2) is one of the growing family of proteins for which well-defined solution and crystal structures have been published and for which small, but distinct differences between these were found. It presents an ideal case to address the question of whether a structural difference is physically real or due to the simplifying approximations with respect to averaging that are used in the conventional methods for structure refinement. NOE distance and (3)J coupling constant restrained molecular dynamics simulations were performed using conventional and time-averaged restraints, both in vacuo and in aqueous solution, and the trajectories were compared with structural properties of published structures. The time-averaged restrained molecular dynamics simulations sampled more conformations at various times and visited states consistent with both previously published solution and crystal structures. It was found that the difference between these structures is due to the refinement methodology used. Application of time-averaged restraints in structure refinement yields a physically different picture of the molecular mobility.