Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein.


Abstract

In vivo selections were used to isolate 43 temperature-sensitive gene V mutants of the bacteriophage f1 from a collection of mutants constructed by saturation mutagenesis of the gene. The sites of temperature-sensitive substitutions are found in both the beta-sheets and the turns of the protein, and some sites are exposed to the solvent while others are not. Thirteen of the variant proteins were purified and characterized to evaluate their free energy changes upon unfolding and their affinities for single-stranded DNA, and eight were tested for their tendencies to aggregate at 42 degrees C. Each of the three temperature-sensitive mutants at buried sites and six of ten at surface sites had free energy changes of unfolding substantially lower (less stabilizing) than the wild-type at 25 degrees C. A seventh mutant at a surface site had a substantially altered unfolding transition and its free energy of unfolding was not estimated. The affinities of the mutant proteins for single-stranded DNA varied considerably, but two mutants at a surface site, Lys69, had much weaker binding to single-stranded DNA than any of the other mutants, while two mutants at another surface site, Glu30, had the highest DNA-binding affinities. The wild-type gene V protein is stable at 42 degrees C, but six of the eight mutants tested aggregated within a few minutes and the remaining two aggregated within 30 minutes at this temperature. Overall, each of the temperature-sensitive proteins tested had a tendency to aggregate at 42 degrees C, and most also had either a low free energy of unfolding (at 25 degrees C), or weak DNA binding. We suggest that any of these properties can lead to a temperature-sensitive gene V phenotype. Study holds ProTherm entries: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 Extra Details: additive : EDTA(1 mM), protein stability; mutagenesis; thermostability; aggregation;,protein-nucleic acid interactions

Submission Details

ID: poNpEXpG3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:14 p.m.

Version: 1

Publication Details
Zabin HB;Terwilliger TC,J. Mol. Biol. (1991) Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein. PMID:2038057
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2GVA 1995-10-15 REFINED SOLUTION STRUCTURE OF THE TYR 41--> HIS MUTANT OF THE M13 GENE V PROTEIN. A COMPARISON WITH THE CRYSTAL STRUCTURE
2GVB 1995-10-15 REFINED SOLUTION STRUCTURE OF THE TYR 41--> HIS MUTANT OF THE M13 GENE V PROTEIN. A COMPARISON WITH THE CRYSTAL STRUCTURE
1GVP 1997-09-04 1.6 GENE V PROTEIN (SINGLE-STRANDED DNA BINDING PROTEIN)
1GKH 1997-09-04 1.7 MUTANT K69H OF GENE V PROTEIN (SINGLE-STRANDED DNA BINDING PROTEIN)
1VQF 1997-02-12 1.8 GENE V PROTEIN MUTANT WITH VAL 35 REPLACED BY ILE 35 AND ILE 47 REPLACED BY VAL 47 (V35I, I47V)
1VQI 1997-02-12 1.8 GENE V PROTEIN MUTANT WITH ILE 47 REPLACED BY VAL 47 (I47V)
1VQA 1997-02-12 1.8 GENE V PROTEIN MUTANT WITH VAL 35 REPLACED BY ALA 35 AND ILE 47 REPLACED BY LEU 47 (V35A, I47L)
1VQC 1997-02-12 1.8 GENE V PROTEIN MUTANT WITH VAL 35 REPLACED BY ILE 35 AND ILE 47 REPLACED BY PHE 47 (V35I, I47F)
1VQE 1997-02-12 1.8 GENE V PROTEIN MUTANT WITH VAL 35 REPLACED BY ILE 35 AND ILE 47 REPLACED BY MET 47 (V35I, I47M)
1VQH 1997-02-12 1.8 GENE V PROTEIN MUTANT WITH ILE 47 REPLACED BY MET 47 (I47M)
1VQB 1997-02-12 1.8 GENE V PROTEIN (SINGLE-STRANDED DNA BINDING PROTEIN)
1VQJ 1997-02-12 1.8 GENE V PROTEIN MUTANT WITH VAL 35 REPLACED BY ILE 35 (V35I)
1VQD 1997-02-12 1.82 GENE V PROTEIN MUTANT WITH VAL 35 REPLACED BY ILE 35 AND ILE 47 REPLACED BY LEU 47 (V35I, I47L)
1VQG 1997-02-12 1.82 GENE V PROTEIN MUTANT WITH ILE 47 REPLACED BY LEU 47 (I47L)
1AE3 1997-09-04 2.0 MUTANT R82C OF GENE V PROTEIN (SINGLE-STRANDED DNA BINDING PROTEIN)
1AE2 1997-09-04 2.0 MUTANT L32R OF GENE V PROTEIN (SINGLE-STRANDED DNA BINDING PROTEIN)
1YHB 1994-06-22 2.2 CRYSTAL STRUCTURES OF Y41H AND Y41F MUTANTS OF GENE V PROTEIN FROM FF PHAGE SUGGEST POSSIBLE PROTEIN-PROTEIN INTERACTIONS IN GVP-SSDNA COMPLEX
2GN5 1986-01-21 2.3 REFINED STRUCTURE OF THE GENE 5 DNA BINDING PROTEIN FROM BACTERIOPHAGE FD
1YHA 1994-06-22 2.5 CRYSTAL STRUCTURES OF Y41H AND Y41F MUTANTS OF GENE V PROTEIN FROM FF PHAGE SUGGEST POSSIBLE PROTEIN-PROTEIN INTERACTIONS IN GVP-SSDNA COMPLEX

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 DNA-Binding protein G5P P69544 G5P_BPM13
100.0 DNA-Binding protein G5P P69542 G5P_BPFD
100.0 DNA-Binding protein G5P P69543 G5P_BPF1