title: Development of a technique for the investigation of folding dynamics of single proteins for extended time periods.
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 ( approximately 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.
authors.affiliation: Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Kinoshita
authors.forename: Kiyoto
authors.initials: K
authors.lastname: Kamagata
authors.forename: Akio
authors.initials: A
authors.lastname: Maeda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Goto
authors.forename: Tamiki
authors.initials: T
authors.lastname: Komatsuzaki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takahashi
mesh_terms: D019411:Clinical Laboratory Techniques / Q000295:instrumentation*; D045304:Cytochromes c / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D038761:Photobleaching; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013997:Time Factors
chemical_list: C014059:CYC1 protein, S cerevisiae; D005456:Fluorescent Dyes; D007527:Isoenzymes; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c