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 estimate intramolecular distances between probes bound to Ca2+-transporting 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'-isothiocyanate. Three different cysteine residues were individually labelled using the following probes: 5-[(2-iodoacetyl)aminoethyl]amino-naphthalene-1-sulfonic acid (I-AEDANS), 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) and fluorescent maleimides. The surface of the membrane was labelled by reconstitution with fluorescent phospholipids (fluorescein and rhodamine derivatives). We found a distance of 4.1 nm from the nucleotide binding site to NBD (at Cys344), and the same distance to fluorescent maleimides (at Cys364). The AEDANS label (at Cys670,672) was found separated 3.5 nm from NBD, 4.4 nm from fluorescent maleimides, and 3.9 nm from the lipid matrix. The NBD label was 3.2 nm apart from fluorescent maleimides and 2.2 nm from the lipid matrix. Finally, fluorescent maleimides were found to be located 4.2 nm above the membrane surface. All these distances agree with a molecular model in which NBD is located in the stalk portion of the Ca2+-ATPase, near the surface of the membrane, and the rest of the probes are above it, in the globular domain of the protein.