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Biophysics
Fluorescence quenching: A tool for single-molecule protein-folding study

Xiaowei Zhuang^*, Taekjip Ha^*,, Harold D. Kim^*, Thomas Centner, Siegfried Labeit^§, and Steven Chu^*,¶

^* Department of Physics, Stanford University, Stanford, CA 94305;  European Molecular Biology Laboratory Heidelberg, Meyerhofstrasse 1, P. O. Box 102209, 69012 Heidelberg, Germany; and ^§ Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164

Contributed by Steven Chu, October 30, 2000

By using titin as a model system, we have demonstrated that fluorescence quenching can be used to study protein folding at the single molecule level. The unfolded titin molecules with multiple dye molecules attached are able to fold to the native state. In the native folded state, the fluorescence from dye molecules is quenched due to the close proximity between the dye molecules. Unfolding of the titin leads to a dramatic increase in the fluorescence intensity. Such a change makes the folded and unfolded states of a single titin molecule clearly distinguishable and allows us to measure the folding dynamics of individual titin molecules in real time. We have also shown that fluorescence quenching can signal folding and unfolding of a small protein with only one immunoglobulin domain.

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