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Mutational Editing of Strained New Protein Folds with Deletion Mutations
Author(s) -
Pattengale Sarah R.,
Kumirov Vlad K.,
Szyszka Taylor N.,
Cordes Matthew H.J.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.598.3
Subject(s) - protein engineering , mutagenesis , biology , dna , microbiology and biotechnology , genetics , chemistry , mutation , biochemistry , gene , enzyme
There are 1,200 known protein folds, some of which evolved from other folds. The consequences and causes of evolutionary fold switching are not well known. The fold switch that occurs in the Cro protein family is an example of new protein fold evolution. There are two major fold groups of Cro proteins: Cro proteins that have an ancestral all‐a fold and those that have a descendant a+b fold. Early forms of the a+b Cro fold, represented by prophage Pfl6 Cro protein, exhibited conformational strain in the turn connecting the first two helices. Two groups of a+b Cro proteins that emerged later show deletions in this turn, either in G14 or T15, with apparent relief of conformational strain. To determine whether a G14 deletion could account for the strain relief, we introduced a G14 deletion into the strained Pfl6 Cro using site‐directed mutagenesis. Using circular dichroism (CD), nuclear magnetic resonance (NMR), and fluorescence anisotropy (FA), we characterized the stability, structure, and DNA binding affinity, respectively, of this Pfl6 variant. The G14 deletion slightly increased the protein's thermal stability, partially relieved the strain in the helix 1–2 turn, and did not significantly change the DNA binding affinity compared to wild‐type Pfl6. These findings suggest that new protein folds may emerge through transitional forms that are edited through deletion and/or insertion mutations to remove awkward features. Support or Funding Information This research is funded by an NIH grant (R01 GM066806).