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

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

^*Materials Sciences Division and ^**Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and ^§Howard Hughes Medical Institute, Department of Chemistry and ^¶Department of Physics, University of California, Berkeley, CA 94720

Contributed by Peter G. Schultz

Abstract

Fluorescence resonance energy transfer and fluorescence polarization anisotropy are used to investigate single molecules of the enzyme staphylococcal nuclease. Intramolecular fluorescence resonance energy transfer and fluorescence polarization anisotropy measurements of fluorescently labeled staphylococcal nuclease molecules reveal distinct patterns of fluctuations that may be attributed to protein conformational dynamics on the millisecond time scale. Intermolecular fluorescence 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.

Footnotes

↵ † Present address: Department of Physics, Stanford University, Stanford, CA 94305.
↵ ‡ T.H. and A.Y.T. contributed equally to this work.
↵ ‖ To whom reprint requests should be addressed. e-mail: pschultz{at}lbl.gov; sweiss{at}lbl.gov.
ABBREVIATIONS:

FRET,

fluorescence resonance energy transfer;

spFRET,

single-pair FRET;

smFPA,

single-molecule fluorescence polarization anisotropy;

SNase,

staphylococcal nuclease;

TMR,

tetramethylrhodamine;

pTp,

deoxythymidine diphosphate;

ssDNA,

single-stranded DNA