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

Masahito Kinoshita, Kiyoto Kamagata, Akio Maeda, Yuji Goto, Tamiki Komatsuzaki, Satoshi Takahashi

Research output: Contribution to journalArticlepeer-review

48 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)10453-10458
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number25
Publication statusPublished - 2007 Jun 19


  • Fluorescence
  • Heterogeneity
  • Iso-1-cytochrome c
  • Unfolded state


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