Characterization of the Tryptophan Fluorescence from Sarcoplasmic Reticulum Adenosinetriphosphatase by Frequency-Domain Fluorescence Spectroscopy

Ignacy Gryczynski, Wieslaw Wiczk, Giuseppe Inesi, Thomas Squier, Joseph R. Lakowicz

Research output: Contribution to journalArticle

42 Scopus citations

Abstract

We examined the tryptophan decay kinetics of sarcoplasmic reticulum Ca2+-ATPase using frequency-domain fluorescence. Consistent with earlier reports on steady-state fluorescence intensity, our intensity decays reveal a reproducible and statistically significant 2% increase in the mean decay time due to calcium binding to specific sites involved in enzyme activation. This Ca2+ effect could not be eliminated with acrylamide quenching, which suggests a global effect of calcium on the Ca2+-ATPase, as opposed to a specific effect on a single water-accessible tryptophan residue. The tryptophan anisotropy decays indicate substantial rapid loss of anisotropy, which can be the result of either intramolecular energy transfer or a change in segmental flexibility of the ATPase protein. Energy transfer from tryptophan to TNP-ATP in the nucleotide binding domain, or to IEADANS on Cys-670 and-674, indicates that most tryptophan residues are 30 A or further away from these sites and that this distance is not decreased by Ca2+. In light of known structural features of the Ca2+-ATPase, the tryptophan fluorescence changes are attributed to stabilization of clustered transmembrane helices resulting from calcium binding.

Original languageEnglish
Pages (from-to)3490-3498
Number of pages9
JournalBiochemistry
Volume28
Issue number8
DOIs
StatePublished - 1 Apr 1989

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