The solution structure of the Tyr41-->His mutant of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13.

The solution structure of mutant Tyr41-->His of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13 has been investigated by nuclear magnetic resonance spectroscopy. Two- and three-dimensional NMR experiments have been employed with a variety of NMR samples of gene V protein, some of which were uniformly enriched with either 15N or 13C. The structure of mutant Tyr41-->His of the M13 gene V protein which occurs in solution as a symmetric dimer was calculated using a two-stage procedure. The first step of the procedure involved the calculation of a set of individual monomer structures using the distance geometry program DIANA. This was then followed by the calculation of dimer structures employing "simulated annealing" protocols with the program X-PLOR. Hereby, the problem of assignment of intra- and inter-subunit NOEs of the symmetric dimer was circumvented through use of a target function that correctly deals with the intra- and inter-subunit contributions to the NOE peaks. Furthermore, a pseudo energy term was employed to restrain the symmetry of the dimer. In addition to this novel calculation strategy, we have incorporated distance information for a set of NOEs which were unambiguously identified as inter-subunit NOEs using an NMR strategy based on asymmetric labelling. A total of 20 structures were calculated for the M13 gene V protein mutant Tyr41-->His based on approximately 1000 experimental restraints derived from the NMR data. The structure of residues 1 to 15 and 29 to 87 of both monomers is reasonably well determined with an average atomic r.m.s. difference between the individual structures and the respective mean structure of approximately 0.9 A for the backbone atoms and approximately 1.4 A for all atoms. The orientation of the exposed anti-parallel beta-loop (residues 16 to 28) with respect to the core could not be determined. The molecular architecture of each of the monomers includes a five-stranded beta-barrel enclosing a hydrophobic core and two-antiparallel beta-loops. The dimer structure is stabilized predominantly by hydrophobic residues primarily involving the symmetry-related dyad domains (residues 64 to 82) of the monomers. Residues which are close to bound single-stranded DNA were identified previously from binding experiments with spin-labelled oligonucleotides. The solution structure of mutant Tyr41-->His of the M13 gene V protein is consistent with these binding data and provides a clear view of the protein's single-stranded DNA binding path.