Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X‐ray crystallography

Abstract

The high-resolution crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, fl, fd) has been solved recently for the wild-type and two surface mutant (Y41F and Y41H) proteins, leading to a plausible model for the polymeric GVP-ssDNA complex (Guan Y, Zhang H, Wang AHJ, 1995, Protein Sci 4187-197). The model of the complex shows extensive contacts between neighboring dimer GVPs involving electrostatic interactions between the K69 from one and the D79 and R82 from the next dimer. In addition, hydrophobic interactions between the amino acids L32 and L44 from one and G23 from the next dimer also contribute to the dimer-dimer interactions. Mutations at the L32, K69, and R82 amino acid sites generally destabilize the protein and many of these affect the function of the phage. We have studied the structural effects of three mutant proteins involving those sites, i.e., L32R, K69H, and R82C, by X-ray crystallographic analysis at 2.0 8, resolution. In L32R GVP, the structural perturbation is localized, whereas in K69H and R82C GVPs, some long-range effects are also detected in addition to the local perturbation. We have interpreted the protein stability and the functional properties associated with those mutations in terms of the observed structural perturbations.

Relative stability of protein structures determined by X‐ray crystallography or NMR spectroscopy: A molecular dynamics simulation study

“…The 39 structures used in this study,9–47 corresponding to 34 different proteins were taken from the Protein Data Bank (PDB) 48. Of these proteins, 26 were identical to those used by Baker and coworkers to test the ROSETTA method for ab initio protein structure prediction 49.…”

A structural double-mutant cycle: estimating the strength of a buried salt bridge in barnase

“…In both RS69 and the double mutant the cavity created by the truncation of the arginine side chain is ®lled by four water molecules. A similar mutation at a dimer±dimer interface of gene V protein (GVP) from Ff phage, Arg823Cys, results in the collapse of a neighbouring side chain into the site of mutation; no water is observed in the created cavity (Su et al, 1997).…”

Structure and function of phage p2 ORF34p2, a new type of single‐stranded DNA binding protein

“…In the ORF34 p2 dimer, the extended, anti‐parallel β‐sheet formed by the one, four and five strands from each monomer observed in Eco SSB is also present and contributes eight hydrogen bonds to the dimer interface. Therefore, the ORF34 p2 dimer is globally similar to that of Eco SSB and different from those formed by other phage SSB proteins such as f1 SSB (Su et al ., 1997), Pf3 SSB (Folmer et al ., 1995) or gp2.5 from coliphage T7 (Hollis et al ., 2001). However, beyond the overall similarity, the differences observed between the ORF34 p2 and the Eco SSB monomers translate into different arrangements of their respective dimers.…”

“…Instead, strands 3 and 4 are connected by an eight residues‐long loop (P50–D57). These features are to some extent present in the structures of SSB proteins from filamentous phages f1 (Su et al ., 1997) and Pf3 (Folmer et al ., 1995). Notwithstanding, ORF34 p2 strongly differs from the f1 SSB and Pf3 SSB proteins with respect to the orientation of the 4′‐5′β‐hairpin, which in ORF34 p2 closely matches that of Eco SSB.…”

“…For these reasons, some phage SSBs have been identified by their sequence similarity to bacterial SSBs whereas others were recognized on the basis of their activity. The crystal structures of phage SSBs, such as the monomeric T4 gp32 (Shamoo et al ., 1995), the dimeric T7 gp2.5 (Hollis et al ., 2001) and GN5 from Escherichia coli filamentous phage f1 (Skinner et al ., 1994; Su et al ., 1997), confirmed their OB‐fold structural and functional motif (Murzin, 1993).…”