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Early folding intermediate of ribonuclease A
1Department of Biochemistry, Beckman Center, Stanford University Medical Center, CA 94305.
Summary
Pulsed hydrogen exchange reveals ribonuclease A rapidly forms a beta-sheet intermediate during folding. This early structure gains stability over time, suggesting cooperative formation and subsequent stabilization by hydrophobic interactions.
Area of Science:
- Protein folding dynamics
- Biophysical characterization of enzymes
Background:
- Ribonuclease A is a model enzyme for studying protein folding.
- Understanding folding intermediates is crucial for elucidating protein structure formation.
Purpose of the Study:
- To characterize the early folding intermediate of ribonuclease A using pulsed hydrogen exchange.
- To investigate the structural properties and stability of the beta-sheet in the folding intermediate.
Main Methods:
- Pulsed hydrogen exchange (2H-1H) was employed to monitor the folding process.
- Proton exchange properties of specific regions (beta-sheet and alpha-helices) were analyzed.
Main Results:
- A principal early folding intermediate (I1) was identified, characterized by rapid beta-sheet formation.
- Hydrogen-bonded probes within the beta-sheet were protected in I1, indicating cooperative formation.
- Initial beta-sheet protection factors increased with folding time, suggesting subsequent stabilization.
- Alpha-helical regions showed less protection and yielded less precise data.
Conclusions:
- The beta-sheet of ribonuclease A forms rapidly and cooperatively early in the folding process.
- The beta-sheet intermediate gains stability over time, likely through hydrophobic interactions.
- The early folding intermediate is not fully populated upon initial formation, deviating from simple sequential folding models.