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Protein folding studied using hydrogen-exchange labeling and two-dimensional NMR
1Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104-6059.
Annual Review of Biophysics and Biomolecular Structure
|January 1, 1992
Summary
Protein folding can occur rapidly, reaching near-native states in milliseconds. Kinetic barriers influence folding pathways, leading to diverse intermediates observed in proteins like cytochrome c, RNase A, and barnase.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Dynamics
Background:
- Protein folding is crucial for biological function.
- Understanding folding pathways, especially rapid folding, remains a challenge.
- Kinetic barriers significantly impact the observation and analysis of folding intermediates.
Purpose of the Study:
- To investigate the kinetics of protein folding using HX-labeling experiments.
- To analyze the role of kinetic barriers in protein folding pathways.
- To characterize distinct folding intermediates observed in different proteins.
Main Methods:
- Hydrogen-deuterium exchange (HX) labeling experiments.
- Utilizing the pH-pulse mode for kinetic analysis.
- Time-resolved analysis of protein folding on millisecond-to-second timescales.
Main Results:
- Protein folding can be remarkably fast, reaching near-native states within milliseconds.
- Kinetic barriers can lead to molecular intermediates or population heterogeneity.
- Observed intermediates include a two-helix intermediate in cytochrome c, a beta-sheet-plus-helix intermediate in RNase A, and a molten globular intermediate in barnase.
Conclusions:
- The observed diversity of folding intermediates reflects variations in kinetic barrier placement.
- Observing kinetic folding intermediates is experimentally challenging, with many intermediates remaining unseen.
- Seen intermediates may represent the cumulative result of multiple preceding folding steps.