title: Frequency-domain fluorescence spectroscopy resolves the location of maleimide-directed spectroscopic probes within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum.
abstract: We have used fluorescence spectroscopy to characterize three covalently bound spectroscopic maleimide derivatives with respect to their location within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum (SR). These derivatives include (1) 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid, (2) 4-(dimethylamino)azobenzene-4'-maleimide, and (3) fluorescein 5'-maleimide. Biochemical assays demonstrate that modification with any of these three derivatives results in the same functional effects, observed following derivatization of cysteines 344 and 364 by N-ethylmaleimide [Saito-Nakatsuka et al. (1987) J. Biochem. (Tokyo) 101, 365-376]. These residues bracket the ATPase's phosphorylation site (Asp 351) and thus may provide spectroscopic probes of the protein's conformation in this essential region. In agreement with sequencing results, SDS-polyacrylamide gels show that maleimide-modified SR exhibits fluorescence exclusively on the A1 tryptic fragment of the Ca-ATPase. Extensive tryptic digestion followed by centrifugation demonstrates essentially all of the fluorescence was associated with the soluble rather than insoluble (membrane-associated) peptides, confirming the predicted extramembranous location of these residues. Utilizing frequency-domain fluorescence spectroscopy, we were able to recover the transient effects associated with a distribution of donor-acceptor distances. We find from these fluorescence resonance energy transfer measurements that covalently bound maleimide probes are 36 A apart, independent of whether a discrete distance is assumed or a distance distribution model is utilized, in which the conformational variability of the protein is taken into account. While a unimodal distance distribution is adequate to describe the intensity decay associated with maleimide-directed donor-acceptor pairs, a bimodal distribution of distances is necessary to describe the frequency response associated with the energy transfer between maleimide-directed chromophores and other covalently bound probes on the Ca-ATPase, consistent with the large spatial separation observed between maleimides. We recover mean distances of 42 and 77 A between maleimide sites and bound FITC (Lys 515) and mean distances of 28 and 37 A between the maleimide- and the iodoacetamide-directed probes (Cys 670 and 674, whose close proximity approximates a single locus). The measured distances are presented in a model and have permitted us to describe a unique arrangement of these covalently bound probes within both the secondary and tertiary structure of the Ca-ATPase. The resolution inherent in the frequency-domain fluorescence technique to multiple donor-acceptor distances should be generally applicable to a wide range of biological systems in which specific labeling of single unique donor-acceptor sites is not feasible.
authors.affiliation: Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
authors.forename: D J
authors.initials: DJ
authors.lastname: Bigelow
authors.forename: G
authors.initials: G
authors.lastname: Inesi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003545:Cysteine; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D008301:Maleimides / Q000378:metabolism* / Q000494:pharmacology; D008433:Mathematics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D008301:Maleimides; D000252:Calcium-Transporting ATPases; D003545:Cysteine