SECONDARY STRUCTURE OF THE SINGLE-STRANDED-DNA BINDING-PROTEIN ENCODED BY FILAMENTOUS PHAGE PF3 AS DETERMINED BY NMR

Nuclear magnetic resonance spectroscopy was employed to study the sing le-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3. The protein is 78 amino acids long and occurs in s olution predominantly as a homodimer with a molecular mass of 18 kDa. Sequence-specific H-1 and N-15 resonance assignments have been obtaine d using homo- and heteronuclear two- and three-dimensional experiments . The secondary structure of the protein monomer was determined from a qualitative interpretation of nuclear Overhauser enhancement spectra and amide exchange data. It consists of a five-stranded antiparallel b eta-sheet and three beta-hairpins. Problems caused by the protein's te ndency to aggregate at concentrations needed for NMR spectroscopy were largely overcome by designing a mutant (Phe36-->His) which exhibits s ignificantly improved solubility characteristics over the wild-type pr otein. It is shown that this mutation only locally affects the structu re of the protein; the chemical shifts of the wild-type and mutant spe cies differ only for a few residues near the site of the mutation, and the secondary structures of the proteins are identical. The secondary structure of the Pf3 single stranded DNA binding protein is compared to that of the Ff gene V protein, the only single-stranded DNA binding protein for which the complete three-dimensional structure is known t o date [Folkers, P. J. M., Nilges, M., Folmer, R. H. A., Konings, R. N . H. and Hilbers, C. W (1994) J. Mol, Biol. 236, 229-246; Skinner, M. M., Zhang, H., Leschnitzer, D. H., Guan, Y., Bellamy, H., Sweet, R. M. , Gray, C. W., Konings, R. N. H., Wang, A, H.-J. and Terwilliger, T. C . (1994) Proc. Natl Acad. Sci. USA 91, 2071-2075]. It is found that th e secondary structures of the two proteins are very similar which supp orts the hypothesis that a five-stranded antiparallel beta-sheet with protruding beta-hairpins is a common motif in a certain class of singl e-stranded DNA binding proteins. In addition, the sequence and folding predicted earlier for the DNA binding wing in the single-stranded DNA binding protein of phage Pf3 [de Jong, E. A. M., van Duynhoven, J. P. M., Harmsen, B. J. M., Tesser, G. I., Konings, R. N. H. and Hilbers, C. W. (1989) J. Mol. Biol. 206, 133-156] is borne out by the present s tudy. It closely resembles that in the single-stranded DNA binding pro tein of phage Ff, which may indicate that such a wing is a recurrent m otif as well.