Yangmee Kim, Dr. James H. Prestegard¹

Department of Chemistry, Yale University, New Haven, Connecticut 06511

1) Correspondence to James H. Prestegard, Department of Chemistry, Yale University, New Haven, CT 06511

Keywords structure determination, two-dimensional NMR, molecular mechanics, molecular dynamics, conformational equilibrium Abstract Structure determination of small proteins using NMR data is most commonly pursued by combining NOE derived distance constraints with inherent constraints based on chemical bonding. Ideally, one would make use of a variety of experimental observation, not just distance constraints. Here, coupling constant constraints have been added to molecular mechanics and molecular dynamics protocols for structure determination in the form of a pseudo-energy function that is minimized in a search for an optimum molecular conformation. Application is made to refinement of a structure for a 77 amino acid protein involved in fatty acid synthesis, Escherichia coli acyl carrier protein (ACP). 54 3JHN coupling constants, 12 coupling constants for stereo-specifically assigned side chain protons, and 450 NOE distance constraints were used to calculate the 3-D structure of ACP. A three-step protocol for a molecular dynamics calculation is described, in analogy to the protocol previously used in molecular mechanics calculations. The structures calculated with the molecular mechanics approach and the molecular dynamics approach using a rigid model for the protein show similar molecular energies and similar agreement with experimental distance and coupling constant constraints. The molecular dynamics approach shows some advantage in overcoming local minimum problems, but only when a two-state averaging model for the protein was used, did molecular energies drop significantly.

Given that XPLOR was developed at Yale in those days, I guess the problems in the structure were caused by XPLOR, but that doesn't mean the authors shouldn't have redone this structure a long time ago. Or, perhaps, given that the exact same structure has been solved many times with X-ray too, the NMR structure should be removed from the PDB and stored in a separate registry where we keep historical remnants.