期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 98, 期 25, 页码 14362-14367出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.191494098
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The global configuration of individual, surface-adsorbed molecules of the giant muscle protein titin, labeled with rhodamine conjugates, was followed with confocal microscopy. Fluorescence-emission intensity was reduced because of self-quenching caused by the close spacing between rhodamine dye molecules that formed dinners. In the presence of chemical denaturants, fluorescence intensity increased, reversibly, up to 5-fold in a fast reaction; the kinetics were followed at the single-molecule level. We show that dinners formed in a concentrated rhodamine solution dissociate when exposed to chemical denaturants. Furthermore, titin denaturation, followed by means of tryptophan fluorescence, is dominated by a slow reaction. Therefore, the rapid fluorescence change of the single molecules reflects the direct action of the denaturants on rhodamine dimers rather than the unfolding/refolding of the protein. Upon acidic denaturation, which we have shown not to dissociate rhodamine dinners, fluorescence intensity change was minimal, suggesting that dinners persist because the unfolded molecule has contracted into a small volume. The highly contractile nature of the acid-unfolded protein molecule derives from a significant increase in chain flexibility. We discuss the potential implications this finding could have for the passive mechanical behavior of striated muscle.
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