Spectral fluctuation of a single fluorophore conjugated to a protein molecule.

Abstract:

:We have measured the fluorescence spectra of a single fluorophore attached to a single protein molecule in aqueous solution using a total internal reflection fluorescence microscope. The most reactive cysteine residue of myosin subfragment-1 (S1) was labeled with tetramethylrhodamine. The spectral shift induced by a change in solvent from aqueous buffer to methanol in both single-molecule and bulk measurements were similar, indicating that, even at the single molecule level, the fluorescence spectrum is sensitive to microenvironmental changes of fluorophores. The time dependence of the fluorescence spectra of fluorophores attached to S1 molecules solely showed a fluctuation with time over a time scale of seconds. Because the fluorescence spectra of the same fluorophores directly conjugated to a glass surface remained constant, the spectral fluctuation observed for the fluorophores attached to S1 is most likely due to slow spontaneous conformational changes in the S1 molecule. Thus, single-molecule fluorescence spectroscopy appears to be a powerful tool to study the dynamic behavior of single biomolecules.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Wazawa T,Ishii Y,Funatsu T,Yanagida T

doi

10.1016/S0006-3495(00)76708-7

subject

Has Abstract

pub_date

2000-03-01 00:00:00

pages

1561-9

issue

3

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(00)76708-7

journal_volume

78

pub_type

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