DIRECT NUCLEAR OVERHAUSER EFFECT REFINEMENT OF CRAMBIN FROM 2-DIMENSIONAL NMR DATA USING A SLOW-COOLING ANNEALING PROTOCOL

The solution structure of crambin has been refined using a direct nuclear Overhauser effect (NOE) simulation approach (DINOSAUR) following a slow-cooling simulated annealing protocol starting from eight previously obtained nmr and the x-ray structures of crambin. Theoretical NOE intensities calculated with inclusion of local motions were directly compared to the experimental nmr data and forces were derived using a simple first-order approximation for the calculation of the NOE gradient. A dynamic assignment procedure was applied for the peaks involving unassigned diastereotopic proton pairs or equivalent aromatic protons. With this approach, R factors could be minimized in a reasonable simulation time to low values (around 0.26) while deviations from ideal bond lengths and angles are still acceptable. The improvement in R factors is accompanied by an improvement of the precision of the structures, the rms deviations (rmsd; from the average) calculated on the ensemble of nine structures decreasing from 0.65 to 0.55 angstrom for backbone atoms and from 1.0 to 0.85 angstrom for all heavy atoms. The solution structure is significantly different from the x-ray structure with rmsd for all atoms of 1.35 angstrom compared to 0.85 angstrom between solution structures. The largest differences are found for residues Thr-21 and Pro-22 in the loop region between the two alpha-helices and for the side chain of Tyr-29. (C) 1994 John Wiley & Sons, Inc.