INTRAMOLECULAR DISTANCES WITHIN THE CA2-ATPASE FROM SARCOPLASMIC-RETICULUM AS ESTIMATED THROUGH FLUORESCENCE ENERGY-TRANSFER BETWEEN PROBES()

Fluorescence energy transfer measurements have been carried out to est imate intramolecular distances between probes bound to Ca2+-transporti ng ATPase (Ca2+-ATPase) as well as distances between these probes and the phospholipid headgroup. The nucleotide binding site was monitored by using 1,N6-ethenoadenosine 5'-triphosphate, a fluorescent analogue of ATP, and also by labelling Lys515 with fluorescein 5'-isothiocyanat e. Three different cysteine residues were individually labelled using the following probes: iodoacetyl)aminoethyl]amino-naphthalene-1-sulfon ic acid (I-AEDANS), 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) and fluorescent maleimides. The surface of the membrane was labelled by r econstitution with fluorescent phospholipids (fluorescein and rhodamin e derivatives). We found a distance of 4.1 nm from the nucleotide bind ing site to NBD (at Cys344), and the same distance to fluorescent male imides (at Cys364). The AEDANS label (at Cys670,672) was found separat ed 3.5 nm from NBD, 4.4 nm from fluorescent maleimides, and 3.9 nm fro m the lipid matrix. The NBD label was 3.2 nm apart from fluorescent ma leimides and 2.2 nm from the lipid matrix. Finally, fluorescent maleim ides were found to be located 4.2 nm above the membrane surface. All t hese distances agree with a molecular model in which NBD is located in the stalk portion of the Ca2+-ATPase, near the surface of the membran e, and the rest of the probes are above it, in the globular domain of the protein.