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Proceedings Paper

Fluorescence quenching in proteins: some applications to protein-DNA and protein-lipid interactions
Author(s): Jay R. Knutson; Raymond F. Chen; D. K. Porter; Preston Hensley; Myun Ki Han; S. J. Kim; Samuel H. Wilson; M. Clague; Cynthia K. Williamson
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Paper Abstract

The authors outline some examples of the advantages found in subdividing overall quenching into heterogeneous contributions. Subdivision is accomplished by overdetermination (global) and association (DAS, decay associated spectral) methods. In some cases, the subdivision of fluorescence leads to the unique identification of different fluorophores in different sites. Alternatively, the recovered components may reflect conformational heterogeneity at each site. For intrinsic protein fluorescence, it is often noted in the literature that single Trp proteins may be multiexponential. Genetic substitution in multi-Trp proteins, however, often leads to very strong (if not complete) lifetime-to-Trp assignment. Even if a single Trp experiences two or more microenvironments, it can be a useful reporter. The linkage of multiple lifetimes and amplitudes to changes in global conformation often reveals a more `sensitive' subpopulation or lifetime component that becomes a better indicator for important conformational states than aggregate intensity can provide. This has proven useful in studying pH transitions of proteins both in solution and embedded in membranes. Energy transfer is particularly useful in differentiating sites at different distances. Further, the disclosure of heterogeneity in distance is clearly superior to the reporting of a mean distance. This report surveys several systems that have been examined via emission DAS techniques, showing how each protein is better understood when viewed in terms of discrete spectral contributions. We conclude with an overview and some details about our construction of an EDAS (excitation-DAS) instrument; i.e., how excitation scans can be incorporated into a time-resolved instrument.

Paper Details

Date Published: 1 April 1992
PDF: 16 pages
Proc. SPIE 1640, Time-Resolved Laser Spectroscopy in Biochemistry III, (1 April 1992); doi: 10.1117/12.58205
Show Author Affiliations
Jay R. Knutson, National Institutes of Health (United States)
Raymond F. Chen, National Institutes of Health (United States)
D. K. Porter, National Institutes of Health (United States)
Preston Hensley, SmithKline Beecham Corp. (United States)
Myun Ki Han, National Institutes of Health (United States)
S. J. Kim, National Cancer Institute (United States)
Samuel H. Wilson, National Cancer Institute (United States)
M. Clague, EMBL (Federal Republic of Germany) (United States)
Cynthia K. Williamson, National Institutes of Health (United States)


Published in SPIE Proceedings Vol. 1640:
Time-Resolved Laser Spectroscopy in Biochemistry III
Joseph R. Lakowicz, Editor(s)

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