Open AccessDevelopment of a technique for the investigation of folding dynamics of single proteins for extended time periods
Author(s) -
Motoi Kinoshita,
Kiyoto Kamagata,
Akio Maeda,
Yuji Goto,
Tamiki Komatsuzaki,
Satoshi Takahashi
Publication year - 2007
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.0700267104
Subject(s) - alexa fluor , fluorescence , folding (dsp implementation) , chemistry , cytochrome c , fluorescence correlation spectroscopy , molecule , biophysics , molecular dynamics , protein folding , analytical chemistry (journal) , chemical physics , crystallography , physics , optics , chromatography , computational chemistry , biochemistry , biology , electrical engineering , organic chemistry , mitochondrion , engineering
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-cytochromec (cytc ). 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. Cytc was labeled with Alexa Fluor 532 at the C-terminal cysteine (cytc -Alexa). In bulk experiments, cytc -Alexa was shown to possess different fluorescence intensity for the native state, the unfolded state (U), and the intermediate state. Single-molecule traces of cytc -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.