Crystal structure, folding, and operator binding of the hyperstable Arc repressor mutant PL8.


Abstract

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 stability 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-A resolution by X-ray crystallography. The overall structure of the mutant is very similar to wild type, but Leu8 makes an additional interstrand hydrogen bond at each end of the beta-sheet of the mutant, increasing the total number of beta-sheet hydrogen bonds from six to eight. Comparison of the refolding and unfolding kinetics of the PL8 mutant and wild-type Arc shows that the enhanced stability of the mutant is accounted for by a decrease in the rate of protein unfolding, suggesting that the mutation acts to stabilize the native state and that the beta-sheet forms after the rate-limiting step in folding. The reduced operator affinity of the PL8 dimer appears to arise because the mutant cannot make the new interstrand hydrogen bonds and simultaneously make the wild-type set of contacts with operator DNA. Study holds ProTherm entries: 889, 890, 891, 892, 893, 894, 13448 Extra Details: additive : EDTA(0.2 mM), Arc repressor; DNA binding affinity; hydrogen bond;,kinetics; stability

Submission Details

ID: Qx3Lzt9K

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:16 p.m.

Version: 1

Publication Details
Schildbach JF;Milla ME;Jeffrey PD;Raumann BE;Sauer RT,Biochemistry (1995) Crystal structure, folding, and operator binding of the hyperstable Arc repressor mutant PL8. PMID:7827088
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