A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate

Neuweiler H, Doose S, Sauer M (2005)
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102(46): 16650-16655.

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The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.
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Neuweiler H, Doose S, Sauer M. A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 2005;102(46):16650-16655.
Neuweiler, H., Doose, S., & Sauer, M. (2005). A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102(46), 16650-16655. doi:10.1073/pnas.0507351102
Neuweiler, H., Doose, S., and Sauer, M. (2005). A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102, 16650-16655.
Neuweiler, H., Doose, S., & Sauer, M., 2005. A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102(46), p 16650-16655.
H. Neuweiler, S. Doose, and M. Sauer, “A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate”, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 102, 2005, pp. 16650-16655.
Neuweiler, H., Doose, S., Sauer, M.: A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 102, 16650-16655 (2005).
Neuweiler, H, Doose, S, and Sauer, Markus. “A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate”. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102.46 (2005): 16650-16655.
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