title: Single-Molecule FRET to Measure Conformational Dynamics of DNA Mismatch Repair Proteins.
abstract: Single-molecule FRET measurements have a unique sensitivity to protein conformational dynamics. The FRET signals can either be interpreted quantitatively to provide estimates of absolute distance in a molecule configuration or can be qualitatively interpreted as distinct states, from which quantitative kinetic schemes for conformational transitions can be deduced. Here we describe methods utilizing single-molecule FRET to reveal the conformational dynamics of the proteins responsible for DNA mismatch repair. Experimental details about the proteins, DNA substrates, fluorescent labeling, and data analysis are included. The complementarity of single molecule and ensemble kinetic methods is discussed as well.
authors: University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.
authors: J W
authors: JW
authors: Gauer
authors: University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.
authors: S
authors: S
authors: LeBlanc
authors: North Carolina State University, Raleigh, NC, United States.
authors: P
authors: P
authors: Hao
authors: North Carolina State University, Raleigh, NC, United States.
authors: R
authors: R
authors: Qiu
authors: Wesleyan University, Middletown, CT, United States.
authors: B C
authors: BC
authors: Case
authors: Wesleyan University, Middletown, CT, United States.
authors: M
authors: M
authors: Sakato
authors: Wesleyan University, Middletown, CT, United States.
authors: M M
authors: MM
authors: Hingorani
authors: University of North Carolina at Chapel Hill, Chapel Hill, NC, United States. Electronic address: derie@email.unc.edu.
authors: D A
authors: DA
authors: Erie
authors: North Carolina State University, Raleigh, NC, United States. Electronic address: krwening@ncsu.edu.
authors: K R
authors: KR
authors: Weninger
mesh_terms: D004247:DNA / Q000737:chemistry; D053843:DNA Mismatch Repair / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D011506:Proteins; D004247:DNA
keywords: ATPase; DNA mismatch repair; Enzyme kinetics; FRET; Single-molecule fluorescence; TIRF microscopy; Transient kinetics; smFRET