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
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:14 p.m.
|Number of data points||52|
|Proteins||DNA-Binding protein G5P ; DNA-Binding protein G5P|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG ; Derived Quantity: ddG|
|Libraries||Mutations for sequence MIKVEIKPSQAQFTTRSGVSRQGKPYSLNEQLCYVDLGNEYPVLVKITLDEGQPAYAPGLYTVHLSSFKVGQFGSLMIDRLRLVPAK|