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Related Experiment Videos

Domains in lambda Cro repressor. A calorimetric study.

Y V Griko1, V V Rogov, P L Privalov

  • 1Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.

Biochemistry
|December 22, 1992
PubMed
Summary

A mutant lambda Cro repressor with a Cys-Cys disulfide bond shows enhanced stability. This cross-link stabilizes both C-terminal and N-terminal domains, impacting protein structure and denaturation behavior.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Thermodynamics

Background:

  • The lambda Cro repressor is a key transcriptional regulator.
  • Understanding its structure-function relationship is crucial for molecular biology.
  • Mutagenesis studies can reveal insights into protein stability and folding.

Purpose of the Study:

  • To investigate the thermodynamic properties of a lambda Cro repressor mutant.
  • To characterize the effect of an introduced disulfide bond on protein stability and structure.
  • To explore the cooperative behavior of different domains within the repressor dimer.

Main Methods:

  • Calorimetry was used to study the thermodynamic properties.
  • Site-directed mutagenesis introduced Cys residues at position 55.
  • Differential scanning calorimetry (DSC) was employed for denaturation studies.
  • Limited proteolysis was used to generate cross-linked fragments.

Main Results:

  • Formation of an S-S cross-link between Cys55 residues stabilized the C-terminal domains into a cooperative unit melting at 110°C.
  • A cross-linked tryptic fragment (residues 22-66) exhibited similar stability.
  • The N-terminal parts, unstable in isolation, formed a cooperative block in the cross-linked dimer, melting 9°C higher than the un-cross-linked protein.
  • Cooperative melting of distant N-terminal segments suggests altered solution structure.

Conclusions:

  • Disulfide cross-linking significantly enhances the stability of the lambda Cro repressor dimer.
  • The Cys55 cross-link induces cooperative behavior in both C-terminal and N-terminal domains.
  • These findings raise new questions about the solution structure and dynamics of the lambda Cro repressor.

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