Abstract
Single-molecule spectroscopy has developed into a widely used method for probing the structure, dynamics, and mechanisms of biomolecular systems, especially in combination with Förster resonance energy transfer (FRET). In this introductory tutorial, essential concepts and methods will be outlined, from the FRET process and the basic considerations for sample preparation and instrumentation to some key elements of data analysis and photon statistics. Different approaches for obtaining dynamic information over a wide range of timescales will be explained and illustrated with examples, including the quantitative analysis of FRET efficiency histograms, correlation spectroscopy, fluorescence trajectories, and microfluidic mixing.
Highlights
Single-molecule spectroscopy has become an integral part of biophysical research and nanobiotechnology, and a wide range of biological questions are addressed with these methods [1], including the mechanisms of molecular machines [2,3,4], protein-nucleic acid interactions [5,6], enzymatic reactions [7,8,9], and protein or RNA folding [10,11], to name but a few
I will briefly summarize the key aspects of single-molecule fluorescence spectroscopy on an introductory level, with a focus on the investigation of protein structure and dynamics with single-molecule Förster resonance energy transfer (FRET)
Distance distributions and relevant timescales The relative magnitudes of the timescales of at least four different processes have an influence on the position and the width of the FRET efficiency histogram: (a) the rotational correlation time of the chromophores, (b) the fluorescence lifetime of the donor, (c) the intramolecular dynamics of the molecule probed by the fluorophores, and (d) the observation timescale
Summary
Single-molecule spectroscopy has become an integral part of biophysical research and nanobiotechnology, and a wide range of biological questions are addressed with these methods [1], including the mechanisms of molecular machines [2,3,4], protein-nucleic acid interactions [5,6], enzymatic reactions [7,8,9], and protein or RNA folding [10,11], to name but a few. Distance distributions and relevant timescales The relative magnitudes of the timescales of at least four different processes have an influence on the position and the width of the FRET efficiency histogram: (a) the rotational correlation time of the chromophores, (b) the fluorescence lifetime of the donor, (c) the intramolecular dynamics of the molecule probed by the fluorophores, and (d) the observation timescale. The presence or absence of a distance distribution will affect the position and shape of the subpopulation peaks in lifetime vs transfer efficiency 2D-histograms (Figure 2d) [13,66,78] These examples illustrate how the timescales of the FRET process and the molecular dynamics influence the transfer efficiencies and their distributions observed in single-molecule experiments. Nonequilibrium dynamics of single molecules Even though kinetic information can often be obtained from equilibrium single-molecule experiments, in many cases it is still essential to probe nonequilibrium
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