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Related Experiment Videos

Engineered Bacillus lentus subtilisins having altered flexibility.

T Graycar1, M Knapp, G Ganshaw

  • 1Genencor International, 925 Page Mill Road, Palo Alto, CA 94304, USA.

Journal of Molecular Biology
|September 24, 1999
PubMed
Summary
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Engineered Bacillus lentus subtilisin variants (RSYSA and DSAI) show altered flexibility at the substrate-binding site, impacting proteolytic activity. This study introduces a novel method for analyzing protein segment mobility changes.

Area of Science:

  • Enzymology
  • Structural Biology
  • Protein Engineering

Background:

  • Bacillus lentus subtilisin is a widely used industrial enzyme.
  • Engineering subtilisin variants can enhance enzymatic activity and alter substrate specificity.
  • Understanding structural changes is key to optimizing enzyme function.

Purpose of the Study:

  • To determine the 3D structures of two engineered Bacillus lentus subtilisin variants (RSYSA and DSAI) with enhanced activity.
  • To develop and apply a novel method for identifying protein segments with altered flexibility.
  • To correlate changes in protein flexibility with observed alterations in proteolytic activity.

Main Methods:

  • X-ray crystallography was used to determine the high-resolution structures of the engineered subtilisin variants.

Related Experiment Videos

  • A statistical analysis of variance (ANOVA) was applied to main-chain temperature factors (B-factors).
  • This method identified significant departures from overall variance to pinpoint regions of altered flexibility.

    • Main Results:

    • The three-dimensional structures of RSYSA and DSAI variants were successfully determined.
    • The analysis revealed significant changes in main-chain mobility in both engineered variants.
    • Residues 125-127 showed increased mobility in RSYSA, while residues 100-104 exhibited decreased mobility in DSAI, both located near the substrate-binding site.
    • Comparison across different crystal forms highlighted the influence of crystal lattice contacts on segment flexibility.

    Conclusions:

    • The engineered subtilisin variants exhibit distinct alterations in protein segment flexibility.
    • These flexibility changes, particularly at the substrate-binding site, are likely responsible for the modified proteolytic activity.
    • The developed method provides a valuable tool for analyzing protein dynamics and relating them to functional changes.