Development of a technique for the investigation of folding dynamics of single proteins for extended time periods

doi:10.1073/pnas.0700267104

Development of a technique for the investigation of folding dynamics of single proteins for extended time periods

*Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan;
^†Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan; and
^‡Nonlinear Sciences Laboratory, Department of Earth and Planetary Sciences, Faculty of Science, Kobe University, Nada, Kobe 657-8501, Japan

Edited by Robert L. Baldwin, Stanford University Medical Center, Stanford, CA, and approved April 25, 2007 (received for review January 13, 2007)

Abstract

A technique was developed for the detection of fluorescence signals from free single molecules for extended time periods and was applied to the characterization of the unfolded states of iso-1-cytochrome c (cyt c). Protein molecules labeled with fluorescent dye were slowly injected into a capillary at concentrations that allow for the observation of one molecule at a time. A laser was introduced into the capillary coaxially, and the fluorescence was imaged as traces by using a lens with a large focal depth and wide field of view. Thus, the traces reflect the time-dependent changes in the fluorescence signals from single proteins. Cyt c was labeled with Alexa Fluor 532 at the C-terminal cysteine (cyt c-Alexa). In bulk experiments, cyt c-Alexa was shown to possess different fluorescence intensity for the native state, the unfolded state (U), and the intermediate state. Single-molecule traces of cyt c-Alexa were recorded by using the device. Intensity histograms of the traces revealed two distributions with broad and narrow widths, which were interpreted to correspond to the U and intermediate state, respectively, observed in the bulk measurements. The broad width of the U suggested the existence of a relatively slow conformational dynamics, which might be consistent with the correlation time (≈15 ms) estimated from the traces assignable to the U. The technique was expected to reveal dynamics of proteins along the folding processes without artifacts caused by immobilization.

Footnotes

^§To whom correspondence may be addressed. E-mail: st{at}protein.osaka-u.ac.jp
Author contributions: M.K., K.K., Y.G., and S.T. designed research; M.K., K.K., A.M., and S.T. performed research; M.K., K.K., and T.K. contributed new reagents/analytic tools; M.K., K.K., A.M., and T.K. analyzed data; and M.K., K.K., Y.G., T.K., and S.T. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0700267104/DC1.
Abbreviations:

Alexa532,

Alexa Fluor 532 C5 maleimide;

cyt c,

iso-1-cytochrome c;

cyt c-Alexa,

cyt c labeled with Alexa532;

EMCCD,

electron-multiplier CCD;

G-Alexa,

glutathione labeled with Alexa532;

Gdm,

guanidium chloride;

N,

native state;

U,

unfolded state;

I,

intermediate state.