Characterization of the tryptophan fluorescence from sarcoplasmic reticulum adenosinetriphosphatase by frequency-domain fluorescence spectroscopy.
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.
Gryczynski
I
I
Department of Biological Chemistry, School of Medicine, University of Maryland, Baltimore 21201.
Wiczk
W
W
Inesi
G
G
Squier
T
T
Lakowicz
J R
JR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004735:Energy Transfer; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium