Localization of site-specific probes on the Ca-ATPase of sarcoplasmic reticulum using fluorescence energy transfer.
Highly reactive sulfhydryls, previously labeled with an iodoacetamide spin label on the Ca-ATPase of sarcoplasmic reticulum, were labeled with the fluorescent probe, 5-(2-[iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), without loss of enzymatic activity. We have selectively measured the apparent distance of the more reactive site, relative to other site-specific probes at both the nucleotide and the high affinity calcium binding sites. Fluorescence energy transfer efficiencies from the donor IAEDANS to two acceptors: fluorescein 5'-isothiocyanate or 2',3'-O-(2,4,3-trinitrophenyl)adenosine monophosphate, situated at or near the nucleotide site, were measured using fluorescence lifetimes and yields. Fluorescence on polyacrylamide gels shows that the IAEDANS and fluorescein 5'-isothiocyanate labels are both associated with the B tryptic fragment. The energy transfer measurements are consistent with distances of 56 and 68 A between IAEDANS and these respective binding sites. On the other hand, energy transfer measurements using the lanthanide, praseodymium (Pr3+), as an acceptor indicate that IAEDANS is located 16-18 A from the binding site(s) of this calcium analog. Pr3+ is shown to be a good analog for calcium binding to the high affinity sites on the enzyme since it competitively displaces calcium, as evidenced by the reversal of the specific calcium-dependent intrinsic fluorescent signal and inactivation of ATPase activity, over the same narrow range in Pr3+ concentration where energy transfer is observed. Our observations suggest that the portion of the B fragment spanning the cytoplasmic portion of the ATPase is folded onto the A fragment, bringing the IAEDANS label in close proximity to the high affinity calcium binding domain.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D066298:In Vitro Techniques; D009282:Naphthalenesulfonates; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds; D013861:Thiocyanates
D000345:Affinity Labels; D005452:Fluoresceins; D009282:Naphthalenesulfonates; D013438:Sulfhydryl Compounds; D013861:Thiocyanates; D000249:Adenosine Monophosphate; C006421:1,5-I-AEDANS; C036744:2',3'-O (2,4,6-trinitrocyclohexadienylidine)adenosine 5'-monophosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate