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 protei ns for which well-defined solution and crystal structures have been pu blished and for which small, but distinct differences between these we re 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 conventi onal methods for structure refinement. NOE distance and (3)J coupling constant restrained molecular dynamics simulations were performed usin g conventional and time-averaged restraints, both in vacuo and in aque ous solution, and the trajectories were compared with structural prope rties of published structures. The time-averaged restrained molecular dynamics simulations sampled more conformations at various times and v isited states consistent with both previously published solution and c rystal structures. It was found that the difference between these stru ctures is due to the refinement methodology used. Application of time- averaged restraints in structure refinement yields a physically differ ent picture of the molecular mobility.