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 single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3. The protein is 78 amino acids long and occurs in solution predominantly as a homodimer with a molecular mass of 18 kDa. Sequence-specific 1H and 15N resonance assignments have been obtained 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 beta-sheet and three beta-hairpins. Problems caused by the protein's tendency to aggregate at concentrations needed for NMR spectroscopy were largely overcome by designing a mutant (Phe36-->His) which exhibits significantly improved solubility characteristics over the wild-type protein. It is shown that this mutation only locally affects the structure of the protein; the chemical shifts of the wild-type and mutant species 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 to date [Folkers, P. J. M., Nilges, M., Folmer, R. H. A., Konings, R. N. H. & 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. & Terwilliger, T. C. (1994) Proc. Natl Acad. Sci. USA 91, 2071-2075]. It is found that the secondary structures of the two proteins are very similar which supports the hypothesis that a five-stranded antiparallel beta-sheet with protruding beta-hairpins is a common motif in a certain class of single-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. & Hilbers, C. W. (1989) J. Mol. Biol. 206, 133-156] is borne out by the present study. It closely resembles that in the single-stranded DNA binding protein of phage Ff, which may indicate that such a wing is a recurrent motif as well.
Folmer
R H
RH
Nijmegen SON Research Center, University of Nijmegen, The Netherlands.
Folkers
P J
PJ
Kaan
A
A
Jonker
A J
AJ
Aelen
J M
JM
Konings
R N
RN
Hilbers
C W
CW
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry*; D006859:Hydrogen; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009587:Nitrogen Isotopes; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D017105:Pseudomonas Phages / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D017931:DNA Primers; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D006859:Hydrogen