CRYSTAL-STRUCTURE, FOLDING, AND OPERATOR BINDING OF THE HYPERSTABLE ARE REPRESSOR MUTANT PL8

Arc repressor is a small, dimeric DNA-binding protein that belongs to the ribbon-helix-helix family of transcription factors. Replacing Pro8 at the N-terminal end of the beta-sheet with leucine increases the st ability of the mutant protein by 2.5 kcal/mol of dimer. However, this enhanced stability is achieved at the expense of significantly reduced DNA binding affinity. The structure of the PL8 mutant dimer has been determined to 2.4-Angstrom resolution by X-ray crystallography. The ov erall structure of the mutant is very similar to wild type, but Leu8 m akes an additional interstrand hydrogen bond at each end of the beta-s heet of the mutant, increasing the total number of beta-sheet hydrogen bonds from six to eight. Comparison of the refolding and unfolding ki netics of the PL8 mutant and wild-type Are shows that the enhanced sta bility of the mutant is accounted for by a decrease in the rate of pro tein unfolding, suggesting that the mutation acts to stabilize the nat ive state and that the beta-sheet forms after the rate-limiting step i n folding. The reduced operator affinity of the PL8 dimer appears to a rise because the mutant cannot make the new interstrand hydrogen bonds and simultaneously make the wild-type set of contacts with operator D NA.