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 Jjournal_title
Biophysical journalauthors
Wazawa T,Ishii Y,Funatsu T,Yanagida Tdoi
10.1016/S0006-3495(00)76708-7subject
Has Abstractpub_date
2000-03-01 00:00:00pages
1561-9issue
3eissn
0006-3495issn
1542-0086pii
S0006-3495(00)76708-7journal_volume
78pub_type
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