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
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:16 p.m.
|Number of data points||19|
|Proteins||Transcriptional repressor arc ; Transcriptional repressor arc ; Transcriptional repressor arc|
|Assays/Quantities/Protocols||Experimental Assay: ddG details:Additives , temp:53.4 C, ionic:KCl,EDTA: 100 M,0.2 mM ; Experimental Assay: m temp:20.0 C ; Experimental Assay: ddG temp:20.0 C, details:Additives EDTA (0.2 mM),, ionic:KCl: 100 M ; Experimental Assay: dG temp:20.0 C ; Experimental Assay: m temp:25.0 C ; Experimental Assay: ddG ionic:KCl: 100 M, details:Additives EDTA (0.2 mM),, temp:25.0 C ; Experimental Assay: dG temp:25.0 C ; Experimental Assay: Tm ; Experimental Assay: dHvH ; Derived Quantity: dTm|
|Libraries||Mutations for sequence MKGMSKMPQFNLRWPREVLDLVRKVAEENGRSVNSEIYQRVMESFKKEGRIGA|