Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives

L. Wang, N. R. Kallenbach

Research output: Contribution to journalArticle

Abstract

Limited cleavage of oxidized and reduced horse heart cytochrome c (Cyt c) and the azide complex of Cyt c by proteinase K at room temperature yields a single cut within the central loop (36-60 in the sequence). Using an assay that allows spectroscopic evaluation of the fraction of intact protein as a function of time, together with a simple kinetic model for proteolysis, fluctuation opening of the loop can be related to the free energy of the corresponding protein. This allows us to estimate quantitatively the free energy difference between the oxidized form of Cyt c and other states using proteolysis as a probe. The results we obtain indicate that oxidized Cyt c is 2.0 kcal mol-1 less stable than the reduced form, and 0.07 kcal mol-1 is more stable than the Cyt c: azide complex at 25 °C. These values agree in magnitude with results from hydrogen exchange and unfolding studies, suggesting that the stability of a protein can be directly related to its structural dynamics.

Original languageEnglish (US)
Pages (from-to)2460-2464
Number of pages5
JournalProtein Science
Volume7
Issue number11
StatePublished - Nov 1998

Fingerprint

Proteolysis
Cytochromes c
Free energy
Derivatives
Azides
Endopeptidase K
Enzyme kinetics
Proteins
Protein Stability
Structural dynamics
Horses
Hydrogen
Assays
Temperature

Keywords

  • Circular dichroism
  • Cytochrome c
  • Free energy
  • Proteinase K
  • Proteolysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives. / Wang, L.; Kallenbach, N. R.

In: Protein Science, Vol. 7, No. 11, 11.1998, p. 2460-2464.

Research output: Contribution to journalArticle

Wang, L. ; Kallenbach, N. R. / Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives. In: Protein Science. 1998 ; Vol. 7, No. 11. pp. 2460-2464.
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