title: Observation of protein folding/unfolding dynamics of ubiquitin trapped in agarose gel by single-molecule FRET.
abstract: A ubiquitin mutant with two Cys mutations, m[C]q/S65C, was site-specifically labeled with two dye molecules, Alexa Fluor 488 (donor) and Alexa Fluor 594 (acceptor), due to the different reactivity of these two Cys residues. This doubly dye-labeled ubiquitin has lower structural stability than wild-type ubiquitin. Taking advantage of this decreased stability, conformational heterogeneity of this protein under nondenaturing condition was observed at the single-molecule level using single-paired Förster resonance energy transfer (FRET) by trapping the protein in agarose gel. Three conformational populations corresponding to folded (E                         (ET) ≈ 0.95), loosely packed (E                         (ET) ≈ 0.72), and unfolded (E                         (ET) ≈ 0.22) structures, and the structural transitions between them were observed. Our results suggest that agarose immobilization is good for observing structural dynamics of proteins under native condition.
authors: Institute of Biological Chemistry, Academia Sinica, No. 128, Sec. 2, Academia Rd, Nankang, Taipei, 115, Taiwan.
authors: Li-Ling
authors: LL
authors: Yang
authors: Michael W-P
authors: MW
authors: Kao
authors: Hsin-Liang
authors: HL
authors: Chen
authors: Tsong-Shin
authors: TS
authors: Lim
authors: Wunshain
authors: W
authors: Fann
authors: Rita P-Y
authors: RP
authors: Chen
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005782:Gels; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D058767:Protein Unfolding; D012685:Sepharose / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D005782:Gels; D055767:Immobilized Proteins; D025801:Ubiquitin; D012685:Sepharose