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Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin
1Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Abstract:
Hydrogen exchange pulse labeling and stopped-flow circular dichroism were used to establish that the structure of the earliest detectable intermediate formed during refolding of apomyoglobin corresponds closely to that of a previously characterized equilibrium molten globule. This compact, cooperatively folded intermediate was formed in less than 5 milliseconds and contained stable, hydrogen-bonded secondary structure localized in the A, G, and H helices and part of the B helix. The remainder of the B helix folded on a much slower time scale, followed by the C and E helices and the CD loop. The data indicate that a molten globule intermediate was formed on the kinetic folding pathway.
Insights
The earliest intermediate in apomyoglobin refolding is a molten globule, forming in under 5 milliseconds. This compact structure contains stable secondary elements in specific helices, preceding slower folding of other regions.
Area of Science:
- Biochemistry and Molecular Biology
- Protein Folding Dynamics
- Biophysical Chemistry
Background:
- Protein folding is crucial for biological function.
- Understanding folding intermediates provides insights into the process.
- Apomyoglobin is a well-studied model protein for folding research.
Purpose of the Study:
- To characterize the structure of the earliest kinetic intermediate in apomyoglobin refolding.
- To determine the folding pathway and timing of secondary structure formation.
- To compare the kinetic intermediate with equilibrium molten globule states.
Main Methods:
- Hydrogen exchange pulse labeling to probe solvent accessibility and structure.
- Stopped-flow circular dichroism to monitor secondary structure changes over time.
- Kinetic analysis of refolding intermediates.
Main Results:
- The earliest detectable intermediate forms in <5 milliseconds.
- This intermediate closely resembles a known equilibrium molten globule.
- Stable secondary structure (A, G, H, part of B helices) forms rapidly.
- Slower folding occurs for the rest of the B helix, C and E helices, and CD loop.
Conclusions:
- A molten globule intermediate is formed on the kinetic folding pathway of apomyoglobin.
- This intermediate possesses cooperatively folded secondary structure.
- The folding pathway involves sequential formation of secondary structural elements over different timescales.