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Related Experiment Videos

Folding pathway mediated by an intramolecular chaperone

U Shinde1, Y Li, S Chatterjee

  • 1Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.

Proceedings of the National Academy of Sciences of the United States of America
|August 1, 1993
PubMed
Summary

The N-terminal propeptide of subtilisin acts as an intramolecular chaperone, essential for enzyme folding. This study identifies a folding-competent state, revealing how residual structure facilitates refolding.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Folding Dynamics

Background:

  • The N-terminal propeptide of subtilisin, a serine protease, is vital for the enzyme's proper folding.
  • This propeptide acts as an intramolecular chaperone, guiding the folding process before being cleaved.
  • Understanding the mechanism of intramolecular chaperone-mediated folding is crucial for protein engineering and biotechnology.

Purpose of the Study:

  • To elucidate the pathway by which intramolecular chaperones mediate protein folding.
  • To identify and characterize the folding-competent state of subtilisin.
  • To determine the role of the N-terminal propeptide in inducing this folding-competent state.

Main Methods:

  • Circular dichroism spectroscopy was employed to analyze acid-denatured subtilisin.

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  • Comparison of folding-competent and folding-incompetent states was performed.
  • Refolding capabilities of subtilisin in the presence and absence of the propeptide were assessed.

    • Main Results:

    • A folding-competent state of subtilisin was identified, capable of refolding to an active conformation without the propeptide.
    • This folding-competent state possesses residual structure absent in folding-incompetent states.
    • The N-terminal propeptide is essential for inducing the formation of this folding-competent state.

    Conclusions:

    • The folding-competent state likely represents an intermediate on the subtilisin folding pathway.
    • The propeptide's role is to facilitate the formation of this crucial intermediate state.
    • Without the propeptide, the formation of the folding-competent state and subsequent refolding are significantly slowed.