| Abstract: |
Arc repressor is a small, dimeric DNA-binding protein that belongs to the ribbon-helixhelix family of transcription factors. Replacing Pro8 at the N-terminal end of the β-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-Å 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 β-sheet of the mutant, increasing the total number of β-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 β-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. © 1995, American Chemical Society. All rights reserved. |
| Keywords: |
dna binding protein; dna-binding proteins; nonhuman; protein stability; structure-activity relationship; crystal structure; hydrogen bond; hydrogen bonding; thermodynamics; crystallography, x-ray; protein structure, tertiary; protein folding; protein secondary structure; repressor protein; repressor proteins; protein structure, secondary; viral proteins; guanidine; guanidines; protein denaturation; heat; operator regions (genetics); priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.
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