Conformational transitions in the calcium adenosinetriphosphatase studied by time-resolved fluorescence resonance energy transfer.
We have used time-resolved fluorescence to study proposed conformational transitions in the Ca-ATPase in skeletal sarcoplasmic reticulum (SR). Resonance energy transfer was used to measure distances between the binding sites of 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (IAEDANS) and fluorescein 5-isothiocyanate (FITC) as a function of conditions proposed to affect the enzyme's conformation. When 1.0 +/- 0.15 IAEDANS is bound per Ca-ATPase, most (76 +/- 4%) of the probes have an excited-state lifetime (tau) of 18.6 +/- 0.5 ns, and the remainder have a lifetime of 2.5 +/- 0.9 ns. When FITC is bound to a specific site on each IAEDANS-labeled enzyme, most of the long-lifetime component is quenched into two short-lifetime components, indicating energy transfer that corresponds to two donor-acceptor distances. About one-third of the quenched population has a lifetime tau = 11.1 +/- 2.5 ns, corresponding to a transfer efficiency E = 0.40 +/- 0.07 and a donor-acceptor distance R1 = 52 +/- 3 A. The remaining two-thirds exhibit lifetimes in the range of 1.2-4.2 ns, corresponding to a second distance 31 A less than or equal to R2 less than or equal to 40 A. Addition of Ca2+ (in the micromolar to millimolar range), or vanadate (to produce a phosphoenzyme analogue), had no effect on the donor-acceptor distances. Addition of decavanadate results in the quenching of IAEDANS fluorescence but has no effect on the energy-transfer distance.(ABSTRACT TRUNCATED AT 250 WORDS)
Birmachu
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W
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Nisswandt
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Thomas
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D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors
D005456:Fluorescent Dyes; D000252:Calcium-Transporting ATPases