A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate
H. Neuweiler, S. Doose, and M. Sauer. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102 (46):
16650-16655(November 2005)
Abstract
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.
%0 Journal Article
%1 Neuweiler2005
%A Neuweiler, H
%A Doose, S
%A Sauer, M
%C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA
%D 2005
%I NATL ACAD SCIENCES
%J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
%K hannes sauer doose
%N 46
%P 16650-16655
%T A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate
%U http://dx.doi.org/10.1073/pnas.0507351102
%V 102
%X 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.
@article{Neuweiler2005,
abstract = {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.},
added-at = {2011-03-01T10:57:13.000+0100},
address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA},
affiliation = {Sauer, M (Reprint Author), Univ Bielefeld, Appl Laser Phys \& Laser Spectroscopy, Univ Str 25, D-33615 Bielefeld, Germany. Univ Bielefeld, Appl Laser Phys \& Laser Spectroscopy, D-33615 Bielefeld, Germany.},
author = {Neuweiler, H and Doose, S and Sauer, M},
author-email = {sauer@physik.uni-bielefeld.de},
biburl = {https://www.bibsonomy.org/bibtex/289794ccc63fa0b068de8d6b4c55a2647/reichert},
doc-delivery-number = {986PT},
groups = {public},
interhash = {34b49a77cfa1c0a7d50384b5a38643e8},
intrahash = {89794ccc63fa0b068de8d6b4c55a2647},
issn = {0027-8424},
journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA},
journal-iso = {Proc. Natl. Acad. Sci. U. S. A.},
keywords = {hannes sauer doose},
keywords-plus = {FLUORESCENCE CORRELATION SPECTROSCOPY; INTRAMOLECULAR CONTACT FORMATION; TRP-CAGE; CONFORMATIONAL DYNAMICS; BINDING PROTEIN; FAST EVENTS; POLYPEPTIDES; SIMULATION; TRYPTOPHAN; KINETICS},
language = {English},
month = {NOV 15},
number = 46,
number-of-cited-references = {38},
pages = {16650-16655},
publisher = {NATL ACAD SCIENCES},
subject-category = {Multidisciplinary Sciences},
times-cited = {65},
timestamp = {2011-03-09T14:16:40.000+0100},
title = {A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate},
type = {Article},
unique-id = {ISI:000233462900024},
url = {http://dx.doi.org/10.1073/pnas.0507351102},
username = {reichert},
volume = 102,
year = 2005
}