REFINED SOLUTION STRUCTURE OF THE TYR41-]HIS MUTANT OF THE M13 GENE-VPROTEIN - A COMPARISON WITH THE CRYSTAL-STRUCTURE

The three-dimensional solution structure of mutant Tyr41-->His of the single-stranded DNA binding protein encoded by gene V of the filamento us bacteriophage M13 has been refined in tale stages. The first stage involved the collection of additional NOE-based distance constraints, which were then used in eight cycles of back-calculations and structur e calculations. The structures of the gene V protein dimers were calcu lated using simulated annealing, employing restrained molecular dynami cs with a geometric force field. In the second stage of the refinement procedure solvent was explicitly included during the dynamic calculat ions. A total of 30 structures was calculated for the protein, represe nting its solution structure in water. The first calculation step sign ificantly improved the convergence of the structures, whereas the subs equent simulations in water made the structures physically more realis tic, This is, for instance, illustrated by the number of hydrogen bond s formed in the molecule, which increased considerably upon going to a queous solution. It is shown that the solution structure of the mutant gene V protein is nearly identical to the crystal structure of the wi ld-type molecule, except for the DNA-binding loop (residues 16-28). Th is antiparallel beta-hairpin is twisted and partially folded back towa rds the core of the protein in the NMR structure, whereas it is more e xtended and points away from the rest of the molecule in the X-ray str ucture. Unrestrained molecular dynamics calculations suggest that this latter conformation is energetically unstable in solution.