Relationship between in vivo activity and in vitro measures of function and stability of a protein.


Abstract

The in vivo activities of mutant proteins are readily measured and can potentially be used to estimate changes in in vitro properties such as stability or function, but this connection has not been rigorously established. Gene V protein is a small protein produced by bacteriophage f1 that binds to single-stranded DNA and to RNA and for which fitness can be assayed both in vivo and in vitro. We have assembled a large number of temperature-sensitive mutants of the gene V protein of bacteriophage f1 and measured their ability to support phage growth and replication in vivo. We have also purified many of these mutant gene V proteins and measured their stabilities and ssDNA binding affinities in vitro. Mutations at surface residues frequently yielded temperature-sensitive mutants, but remarkably, no overall correlation between in vivo activity and in vitro measures of either stability or function was found for this group. Mutations at buried residues often lead to the temperature-sensitive phenotype. At buried sites temperature sensitivity was strongly correlated with in vitro stability changes, but not with in vitro ssDNA binding affinity. The implication of these observations for protein engineering efforts is that phenotypes conferred by amino acid substitutions at buried sites can be used to identify mutants whose stabilities fall into ranges of interest, while phenotypes of mutants with surface substitutions may be much less readily interpreted, even in the case of a single-stranded-DNA-binding protein. Study holds ProTherm entries: 3228, 3229, 3230, 3231, 3232, 3233, 3234, 3235, 3236, 3237, 3238, 3239, 3240, 3241, 3242, 3243, 3244, 3245, 3246, 3247, 3248, 3249, 3250, 3251, 3252, 3253, 3254, 3255, 3256, 3257, 3258, 3259, 3260, 3261, 3262, 3263, 3264, 3265, 3266, 3267, 3268, 3269, 3270, 3271, 3272, 3273, 3274, 3275, 3276, 3277, 3278, 3279, 3280, 3281, 3282, 3283, 3284, 3285, 3286, 3287, 3288, 3289 Extra Details: additive : EDTA(1 mM),ddG values were measured in the presence of 2M GdnHCl gene V protein, bacteriophage f1, combinatorial mutagenesis;,protein stability; DNA binding affinity

Submission Details

ID: mhic2h4r3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:21 p.m.

Version: 1

Publication Details
Sandberg WS;Schlunk PM;Zabin HB;Terwilliger TC,Biochemistry (1995) Relationship between in vivo activity and in vitro measures of function and stability of a protein. PMID:7547934
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
2GN5 1986-01-13T00:00:00+0000 2.3 REFINED STRUCTURE OF THE GENE 5 DNA BINDING PROTEIN FROM BACTERIOPHAGE FD
2GVA 1995-07-27T00:00:00+0000 0 REFINED SOLUTION STRUCTURE OF THE TYR 41--> HIS MUTANT OF THE M13 GENE V PROTEIN. A COMPARISON WITH THE CRYSTAL STRUCTURE
2GVB 1995-07-27T00:00:00+0000 0 REFINED SOLUTION STRUCTURE OF THE TYR 41--> HIS MUTANT OF THE M13 GENE V PROTEIN. A COMPARISON WITH THE CRYSTAL STRUCTURE
1AE2 1997-03-04T00:00:00+0000 2.0 MUTANT L32R OF GENE V PROTEIN (SINGLE-STRANDED DNA BINDING PROTEIN)
1AE3 1997-03-04T00:00:00+0000 2.0 MUTANT R82C OF GENE V PROTEIN (SINGLE-STRANDED DNA BINDING PROTEIN)
1GKH 1997-03-04T00:00:00+0000 1.7 MUTANT K69H OF GENE V PROTEIN (SINGLE-STRANDED DNA BINDING PROTEIN)
1GVP 1997-02-26T00:00:00+0000 1.6 GENE V PROTEIN (SINGLE-STRANDED DNA BINDING PROTEIN)
1VQA 1996-08-14T00:00:00+0000 1.8 GENE V PROTEIN MUTANT WITH VAL 35 REPLACED BY ALA 35 AND ILE 47 REPLACED BY LEU 47 (V35A, I47L)
1VQB 1996-08-14T00:00:00+0000 1.8 GENE V PROTEIN (SINGLE-STRANDED DNA BINDING PROTEIN)
1VQC 1996-08-14T00:00:00+0000 1.8 GENE V PROTEIN MUTANT WITH VAL 35 REPLACED BY ILE 35 AND ILE 47 REPLACED BY PHE 47 (V35I, I47F)

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