LOCALIZED SOLUTION STRUCTURE REFINEMENT OF AN F45W VARIANT OF UBIQUITIN USING STOCHASTIC BOUNDARY MOLECULAR-DYNAMICS AND NMR DISTANCE RESTRAINTS

The local structure within an 8-Angstrom radius around residue 45 of a recombinant F45W variant of human ubiquitin has been determined using 67 interproton distance restraints measured by two-dimensional proton NMR. Proton chemical shift evidence indicates that structural perturb ations due to the F45W mutation are minimal and limited to the immedia te vicinity of the site of mutation. Simulated annealing implemented w ith stochastic boundary molecular dynamics was applied to refine the s tructure of Trp 45 and 10 neighboring residues. The stochastic boundar y method allowed the entire protein to be reassembled from the refined coordinates and the outlying unrefined coordinates with little distor tion at the boundary. Refinement began with four low-energy indole rin g orientations of F45W-substituted wild-type (WT) ubiquitin crystal co ordinates. Distance restraints were derived from mostly long-range NOE cross peaks with 51 restraints involving the Trp 45 indole ring. Tand em refinements of 64 structures were done using either (1) upper and l ower bounds derived from qualitative inspection of NOE cross-peak inte nsities or (2) quantitative analysis of cross-peak heights using the p rogram MARDIGRAS. Though similar to those based on qualitative restrai nt, structures obtained using quantitative NOE analysis were superior in terms of precision and accuracy as measured by back-calculated sixt h-root R factors. The six-membered portion of the indole ring is nearl y coincident with the phenyl ring of the WT and the indole NH is expos ed to solvent. Accommodation of the larger ring is accompanied by smal l perturbations in the backbone and a 120 degrees rotation of the chi( 2) dihedral angle of Leu 50.