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.

Keywords: ATPase; DNA mismatch repair; Enzyme kinetics; FRET; Single-molecule fluorescence; TIRF microscopy; Transient kinetics; smFRET.