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Proceedings Paper

Applications of room-temperature tryptophan phosphorescence to the study of protein structure and dynamics
Author(s): Joseph V. Mersol; Anne Gershenson; Duncan G. Steel; Ari Gafni
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Paper Abstract

Most proteins are capable of emitting tryptophan phosphorescence at room temperature in deoxygenated aqueous solutions. Like fluorescence, phosphorescence intensities and lifetimes are useful for studying protein structure. Phosphorescence differs from fluorescence, however, in several ways. Phosphorescence occurs on a time-scale of msec-sec, while fluorescence decays in nanoseconds. Second, the phosphorescence decay of a single tryptophan is nearly monoexponential, making assignments of decay components to individual residues possible. Finally, phosphorescence is a more sensitive probe of the local tryptophan environment, as the lifetime can change by orders of magnitude depending on site rigidity and other factors. The authors describe applications of phosphorescence spectroscopy for protein study. In particular, tryptophan phosphorescence quenching by resonance energy transfer to freely diffusing acceptors was used to show that Trp 109 is the origin of phosphorescence in E. coli alkaline phosphatase (AP). By following changes in the emissive lifetime of this deeply buried residue, the presence of an enzymatically active but structurally modified intermediate state is detected in the unfolding of AP in high concentrations of Guanidine:HCl, and followed the kinetics of the decline in activity upon further unfolding. In addition to the new understanding of AP, the results of these experiments show that room temperature tryptophan phosphorescence is a powerful tool for the study of proteins.

Paper Details

Date Published: 1 April 1992
PDF: 11 pages
Proc. SPIE 1640, Time-Resolved Laser Spectroscopy in Biochemistry III, (1 April 1992); doi: 10.1117/12.58238
Show Author Affiliations
Joseph V. Mersol, Univ. of Michigan (United States)
Anne Gershenson, Univ. of Michigan (United States)
Duncan G. Steel, Univ. of Michigan (United States)
Ari Gafni, Univ. of Michigan (United States)

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

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