Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism

Abstract

Fluorescence resonance energy transfer and f luorescence polarization anisotropy are used to investigate single molecules of the enzyme staphylococcal nuclease. Intramolecular f luorescence resonance energy transfer and f luorescence polarization anisotropy measurements of f luorescently labeled staphylococcal nuclease molecules reveal distinct patterns of f luctuations that may be attributed to protein conformational dynamics on the millisecond time scale. Intermolecular f luorescence resonance energy transfer measurements provide information about the dynamic interactions of staphylococcal nuclease with single substrate molecules. The experimental methods demonstrated here should prove generally useful in studies of protein folding and enzyme catalysis at single-molecule resolution.Single-molecule spectroscopy can provide information about complex biological molecules and systems that is difficult to obtain from ensemble measurements (1-6). For example, one can observe the time trajectories of single molecules in biochemical reactions that cannot be synchronized in ensemble experiments. In a population of molecules heterogeneous in a particular property, single-molecule spectroscopy can also resolve and quantitatively compare distinct subpopulations that would be indistinguishable at the ensemble level.Fluorescence resonance energy transfer (FRET) measurements between single pairs of acceptor and donor fluorophores can yield information about structural relationships and distance fluctuations between regions of a single biomolecule or between components of an interacting system of biomolecules (7-10). In addition, the rotational dynamics of a single fluorophore can be probed by monitoring fluctuations in fluorescence polarization (11)(12)(13)(14). Here we develop the techniques of single-pair FRET (spFRET) and singlemolecule fluorescence polarization anisotropy (smFPA) and show how they can be used to observe the conformational fluctuations and catalytic reactions of enzymes at singlemolecule resolution.Staphylococcal nuclease (SNase) is a 19 kDa Ca 2ϩ -dependent enzyme that catalyzes the hydrolysis of DNA and RNA into mono-and dinucleotides (15). Its catalytic mechanism, thermodynamic stability, and folding pathway have been studied extensively at the ensemble level (16-21). To probe the conformational dynamics of SNase and its interactions with substrate at single-molecule resolution, three experimental methods were used. First, intramolecular spFRET was measured between donor and acceptor fluorophores attached to single SNase proteins. Second, single-molecule fluorescence polarization anisotropy measurements were performed by using SNase labeled singly with one type of fluorophore. Third, intermolecular spFRET was measured between donor-labeled SNase and acceptor-labeled DNA substrate.Using intramolecular spFRET measurements on single SNase protein molecules, we observe interesting dynamics including gradual fluctuations in the FRET efficiencies. A combination of smFPA measurements, simulations, and...

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

“…To analyze the time trajectories of fluorescence signal from a molecule, we first filtered out blinking events (defined as a reversible transition of the acceptor to an inactive state, giving rise to an unquenched donor emission) [29] and photobleaching of either fluorophore, and then performed an analysis on the efficiency of FRET (E app ) time traces. E app here is taken to be the measured fluorescence signal from the acceptor divided by the sum of fluorescence signals from both the acceptor and the donor.…”

“…A scanning confocal fluorescence microscope (SCFM) that is sensitive enough to detect single fluorescent molecules can be used to observe single-pair fluorescence resonance energy transfer (spFRET) [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. Fluorescence resonance energy transfer (FRET) is a spectroscopic process in which non-radiative energy transfer occurs between an excited dipole (the donor) and another dipole (the acceptor) that has an absorption spectrum that overlaps the emission spectrum of the donor [46].…”

“…Changes in the conformation of the molecule are detected as changes in the energy transfer between the donor and acceptor molecule. In this fashion, single molecular-pair FRET has been used to study, e.g., nanometer conformational motions in individual RNA molecules [29][30][31][32][33][34][35], individual DNA molecules [36][37][38][39][40][41] and individual helicases and other motor proteins on DNA [42][43][44][45]. By following changes in FRET efficiency over time, it is possible to observe nanometer distance changes due to macromolecular rearrangements.…”

Multipoint Fluorescence Quenching-Time Statistics for Single Molecules with Anomalous Diffusion

“…FRET has recently been used to probe conformational dynamics of staphylococcal nuclease (SNase) and catalytic turnovers of DNA and RNA hydrolysis into mono-and dinucleotides. 16,21,22,25 Both discrete-jump and continuous models have been used in recent studies of dynamically fluctuating environments of a single molecule. Xie and co-workers have studied the distribution of jump rates by following the closing and opening of single-stranded DNA hairpins and observed multiexponential decay of the two-time correlation functions of fluorescence lifetime fluctuation.…”

“…15,[18][19][20] Fluorescence resonance energy transfer (FRET) measurements between single pairs of donor and acceptor fluorophores provide information about structure and distance fluctuations of a single biomolecule (RNA, DNA, enzymes) or between components of interacting biomolecules (e.g., lifetime of an enzymesubstrate contact, opening and closing of ion channels in a membrane). [21][22][23][24] FRET data reflect conformational states in the molecular center-of-mass frame and are not complicated by overall translocations or rotations. FRET measurements on a single molecule can access conformational subpopulations and dynamics, ligand binding, kinetics of folding/unfolding and protein aggregation, and enzyme catalysis.…”