title: Significant Heterogeneity and Slow Dynamics of the Unfolded Ubiquitin Detected by the Line Confocal Method of Single-Molecule Fluorescence Spectroscopy.
abstract: The conformation and dynamics of the unfolded state of ubiquitin doubly labeled regiospecifically with Alexa488 and Alexa647 were investigated using single-molecule fluorescence spectroscopy. The line confocal fluorescence detection system combined with the rapid sample flow enabled the characterization of unfolded proteins at the improved structural and temporal resolutions compared to the conventional single-molecule methods. In the initial stage of the current investigation, however, the single-molecule Förster resonance energy transfer (sm-FRET) data of the labeled ubiquitin were flawed by artifacts caused by the adsorption of samples to the surfaces of the fused-silica flow chip and the sample delivery system. The covalent coating of 2-methacryloyloxyethyl phosphorylcholine polymer to the flow chip surface was found to suppress the artifacts. The sm-FRET measurements based on the coated flow chip demonstrated that the histogram of the sm-FRET efficiencies of ubiquitin at the native condition were narrowly distributed, which is comparable to the probability density function (PDF) expected from the shot noise, demonstrating the structural homogeneity of the native state. In contrast, the histogram of the sm-FRET efficiencies of the unfolded ubiquitin obtained at a time resolution of 100 μs was distributed significantly more broadly than the PDF expected from the shot noise, demonstrating the heterogeneity of the unfolded state conformation. The variety of the sm-FRET efficiencies of the unfolded state remained even after evaluating the moving average of traces with a window size of 1 ms, suggesting that conformational averaging of the heterogeneous conformations mostly occurs in the time domain slower than 1 ms. Local structural heterogeneity around the labeled fluorophores was inferred as the cause of the structural heterogeneity. The heterogeneity and slow dynamics revealed by the line confocal tracking of sm-FRET might be common properties of the unfolded proteins.
authors.affiliation: Institute for Multidisciplinary Research for Advanced Materials, Tohoku University , Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.|Department of Chemistry, Graduate School and Faculty of Science, Tohoku University , Sendai 980-8578, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Saito
authors.affiliation: Institute for Multidisciplinary Research for Advanced Materials, Tohoku University , Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.|Department of Chemistry, Graduate School and Faculty of Science, Tohoku University , Sendai 980-8578, Japan.
authors.forename: Supawich
authors.initials: S
authors.lastname: Kamonprasertsuk
authors.affiliation: Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University , Sendai 981-8555, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Suzuki
authors.affiliation: Department of Microbial Resources, Graduate School of Agricultural Science, Tohoku University , Sendai 981-8555, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Nanatani
authors.affiliation: Institute for Multidisciplinary Research for Advanced Materials, Tohoku University , Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.|Department of Chemistry, Graduate School and Faculty of Science, Tohoku University , Sendai 980-8578, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Oikawa
authors.affiliation: Department of Bioengineering, The University of Tokyo , 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Kushiro
authors.affiliation: Department of Bioengineering, The University of Tokyo , 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Madoka
authors.initials: M
authors.lastname: Takai
authors.affiliation: Institute of Biological Chemistry, Academia Sinica , No. 128, Sec. 2, Academia Rd, Nankang, Taipei 11529, Taiwan.|Institute of Biochemical Sciences, National Taiwan University , No. 1. Sec. 4, Roosevelt Rd, Taipei 10617, Taiwan.
authors.forename: Po-Ting
authors.initials: PT
authors.lastname: Chen
authors.affiliation: Institute of Biological Chemistry, Academia Sinica , No. 128, Sec. 2, Academia Rd, Nankang, Taipei 11529, Taiwan.
authors.forename: Eric H-L
authors.initials: EH
authors.lastname: Chen
authors.affiliation: Institute of Biological Chemistry, Academia Sinica , No. 128, Sec. 2, Academia Rd, Nankang, Taipei 11529, Taiwan.|Institute of Biochemical Sciences, National Taiwan University , No. 1. Sec. 4, Roosevelt Rd, Taipei 10617, Taiwan.
authors.forename: Rita P-Y
authors.initials: RP
authors.lastname: Chen
authors.affiliation: Institute for Multidisciplinary Research for Advanced Materials, Tohoku University , Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.|Department of Chemistry, Graduate School and Faculty of Science, Tohoku University , Sendai 980-8578, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takahashi
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D011108:Polymers / Q000737:chemistry; D011336:Probability; D058767:Protein Unfolding; D000072760:Single Molecule Imaging; D013816:Thermodynamics; D025801:Ubiquitin / Q000032:analysis* / Q000302:isolation & purification
chemical_list: D011108:Polymers; D025801:Ubiquitin