Sequence‐specific ¹H‐NMR assignment and secondary structure of the Tyr41 → His mutant of the single‐stranded DNA binding protein, gene V protein, encoded by the filamentous bacteriophage M13

Abstract

Sequence-specific 'H-NMR assignments are reported for the Tyr41 --f His (Y41H) mutant of the singlestranded DNA binding protein, encoded by gene V of the filamentous bacteriophage MI3 (GVP). The mutant protein was chosen for this purpose because it exhibits significantly improved solubility characteristics over wildtype GVP [Folkers et al. (1991) Eur. J . Biochem. 200, 139-1481. The secondary structure elements present in the protein are deduced from a qualitative interpretation of the nuclear Overhauser enhancement spectra and amide exchange data. The protein is entirely composed of antiparallel P-structure. It is shown that identical structural elements are present in wild-type GVP. Previously, we have demonstrated that the secondary structure of the flloop, encompassing residues 13-31 which is present in GVP in solution, deviates from that proposed for the same amino acid sequence on the basis of X-ray diffraction data [van Duynhoven et al. (1990) FEBS Lett. 261, 1-41, Now that we have arrived at a complete description of the secondary structure of the protein in solution, other deviations with respect to the crystallographically determined structure became apparent as well. The Nterminal part of the protein is, in solution, part of a triple-stranded P-sheet while, in the crystal, it is an extended strand pointing away from the bulk of the protein dimer. One of the antiparallel P-sheets in the protein which had been designated earlier as the complex loop has, in the solution structure, a different pairwise arrangement of the residues in its respective P-ladders. Residues 30 and 48 are opposite to one another in the solution structure while in the crystal structure residues 32 and 48 are paired. A similar observation is made for the so-called dyad domain of the protein of which the &sheet in the solution structure is shifted by one residue with respect to that of the crystal structure.The genomes of the filamentous bacteriophages containing single-stranded DNA (ssDNA), such as Ff(M13, fd and fl), IKe and Pf3, code for an ssDNA binding protein which plays an indispensible role in the phage replication process [I]. Late in infection, the ssDNA binding protein encoded by gene V (GVP) causes the transition from double-stranded DNA replication to the asymmetric synthesis of the viral strand by forming a tight complex with it. Furthermore, GVP negatively regulates, at the level of translation, the synthesis of a number of phage-encoded proteins including the DNA replication proteins encoded by the genes I1 and X and that of its own [2 -51. Since it was first isolated, the protein has been subjected to various physico-chemical studies and it is considered an important model for protein -ssDNA interactions. The GVP encoded by the filamentous phage Ff(M13, fd and f l ) consists of 87 amino acids, and in solution occurs predominantly as a dimer with a molecular mass of 19.4 kDa. It is the only ssDNA Ahhreviurions. GVP, gene V protein; GVP Y41H, GVP with Tyr41 replaced by His; ssDNA, single-stranded DNA, TOCSY, total cor...

Secondary Structure of the Single‐stranded DNA Binding Protein Encoded by Filamentous Phage Pf3 as Determined by NMR

“…However, all amide protons in this strand exchange rapidly. This suggests a certain degree of flexibility in this p-loop, a phenomenon also observed in the NMR analysis of a similar loop in the ssDNA binding proteins of the phages Ff and IKe (van Duynhoven et al, 1992;Folkers et al, 1991b).…”

“…Furthermore, the amide proton of Arg65 exchanges slowly, which points to yet another hydrogen bond, probably with the carbonyl of Gln41. Based on the secondary and tertiary structure of the Ff ssDNA binding protein (Folkers et al, 1991b, we believe that these contacts are intermonomeric, i.e. the Gln41 and Arg65 just mentioned are members of different monomers.…”

“…A similar mutation proved to be extremely useful in NMR studies on the Ff GVP (Folkers et al, 1991a,b), eventually leading to elucidation of its structure . The secondary structure of the Pf3 ssDBP is presented as well and compared with that of Ff GVP (Folkers et al, 1991b). The secondary structure of the Ff GVP is identical to that of the gene V protein encoded by bacteriophage IKe (van Duynhoven et al, 1992); they are the only two ssDNA binding proteins for which the secondary structures have been published to date.…”

Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids.

“…From this core protrude two prominent 1-hairpins and a large loop. The hairpins are composed of residues 11-25 and 57-70 and are designated the 'DNA binding wing' and 'dyad loop' respectively (Folkers et al, 1991b;Folmer et al, 1994a). The large loop consists of residues 31-36 and is referred to as the 'complex loop'.…”

Sequence‐structure mapping errors in the PDB: OB‐fold domains

“…Unlike the earlier cases, the 2.3 Å resolution structure of the gene 5 DNA binding protein from bacteriophage fd (2gn5; Brayer and McPherson 1983) has been suspected to have problems in a number of previous reports. Both the structure quality assessment methods (Morris et al 1992; Sippl 1993) and a comparison with the results of an NMR study (Folkers et al 1991) pointed to the presence of structural flaws in 2gn5. This is not surprising in the view of massive sequence register errors present in this structure.…”