Work

Characterization of the tryptophan fluorescence from sarcoplasmic reticulum adenosine triphosphatase by frequency-domain fluorescence spectroscopy

Year: 1989

Type: article

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.

Source: Biochemistry

DOI: 10.1021/bi00434a051

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

Institution: University of Maryland, Baltimore

Language: English

Topic: ATP Synthase and ATPases Research

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: National Heart, Lung, and Blood Institute, National Institute of General Medical Sciences, National Center for Research Resources

Awards: G1965936037, G5019390365, Center for Fluorescence Spectroscopy