Tolerance of a protein to multiple polar-to-hydrophobic surface substitutions.


Abstract

Hydrophobic substitutions at solvent-exposed positions in two alpha-helical regions of the bacteriophage P22 Arc repressor were introduced by combinatorial mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were poorly tolerated as shown by the finding that mutants with more than two additional hydrophobic residues were biologically inactive. Several inactive helix A variants were purified and found to have reduced thermal stability relative to wild-type Arc, with a rough correlation between the number of polar-to-hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as many as five polar-to-hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a t(m) 6 degrees C higher than wild-type. By contrast, a helix A mutant with three similar polar-to-hydrophobic substitutions was 23 degrees C less stable than wild-type. Also, one set of three polar-to-hydrophobic substitutions in helix B was tolerated when introduced into the wild-type background but not when introduced into an equally active mutant having a nearly identical structure. Context effects occur both when comparing different regions of the same protein and when comparing the same region in two different homologues. Study holds ProTherm entries: 5457, 5458 Extra Details: additive : EDTA(0.2 mM), aggregation; Arc repressor; combinatorial mutagenesis;,denatured state; protein folding; thermal stability

Submission Details

ID: n4f89U4o3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details
Cordes MH;Sauer RT,Protein Sci. (1999) Tolerance of a protein to multiple polar-to-hydrophobic surface substitutions. PMID:10048325
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