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Related Experiment Videos

Protein conformer selection by ligand binding observed with crystallography

Y Cao1, R A Musah, S K Wilcox

  • 1Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

Protein Science : a Publication of the Protein Society
|March 26, 1998
PubMed
Summary

Cytochrome c peroxidase (CCP) undergoes large conformational changes, shifting between closed and open states. This protein dynamics study reveals a two-step ligand binding mechanism involving an open protein conformation.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Dynamics

Background:

  • Cytochrome c peroxidase (CCP) is crucial for cellular redox homeostasis.
  • Understanding protein conformational dynamics is key to elucidating enzyme mechanisms.
  • Ligand binding can induce significant structural rearrangements in proteins.

Purpose of the Study:

  • To directly observe and characterize large-scale conformational changes in CCP.
  • To investigate the role of protein dynamics in the ligand binding mechanism of CCP.
  • To determine the population of different conformational states in solution.

Main Methods:

  • Protein crystallography was employed to trap and visualize individual protein conformations.
  • Exogenous ligand binding was used to induce and stabilize distinct structural states.

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  • Solution kinetics were utilized to characterize the binding mechanism and transition rates.

    • Main Results:

    • A large-scale conformational change involving the Pro190-Asn195 loop of CCP was directly observed.
    • Ligand binding displaced a buried indole ring (Trp191), exposing it to the solvent.
    • Kinetic data support a two-step binding mechanism where protein opening is rate-limiting.
    • Approximately 4% of wild-type CCP exists in the open conformation in solution, independent of the ligand.

    Conclusions:

    • CCP exhibits significant conformational flexibility, readily transitioning between closed and open states.
    • The observed conformational change is a prerequisite for ligand binding, indicating a dynamic allosteric mechanism.
    • The study provides direct evidence for functionally relevant protein dynamics in CCP, influencing its catalytic activity.