Contributions of chemical derivatization and spectroscopic studies to the characterization of the Ca2+ transport ATPase of sarcoplasmic reticulum.
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Amino Acid Sequence↗
Biomedical subjects
D J Bigelow
Publications and source records attributed to D J Bigelow.
Phosphorylation-dependent changes in the spatial relationship between Ca-ATPase polypeptide chains in sarcoplasmic reticulum membranes.
In order to investigate possible structural changes associated with the coupling mechanisms of the Ca-ATPase in sarcoplasmic reticulum membranes, we have utilized fluorescence resonance energy transfer between spectroscopic probes covalently bound to different domains of the ATPase. Using time-correlated single photon counting, we have directly measured the energy transfer efficiency between 5-[2-[(iodoacetyl)amino]ethyl]aminonaphthalene-1-sulfonic acid (IAEDANS), that is specifically bound to the B trypic fragment at cysteines 670 and 674 and acceptors covalently bound either near the nucleotide binding site, i.e. fluorescein 5-isothiocyanate at lysine 515, also on the B fragment, or maleimide-directed probes specifically located on the A1, tryptic fragment, i.e. 4-dimethylaminoazobenzene-4'-maleimide (DABmal) or fluorescein-5-maleimide (Fmal), probably at cysteines 344 and 364. All of these donor-acceptor pairs exhibit energy transfer both within and between Ca-ATPase molecules allowing us to investigate spatial relationships between the A1 and B domains and between different ATPase polypeptide chains. Differentiation between the intra- and intermolecular components of energy transfer was accomplished in two ways: 1) by comparing the transfer efficiencies in native membranes before and after detergent solubilization and 2) by reconstituting ATPase chains that have already been labeled with either the donor or acceptor chromophores. Using this approach, we find no significant change in the intramolecular transfer efficiency between any of these donor-acceptor pairs either upon binding of calcium to the high affinity sites or upon stabilization of the phosphoenzyme intermediate, indicating that there are no large structural changes within the B tryptic fragment or, alternatively, between the A1 and B fragments. With respect to intermolecular energy transfer, we observe no effect of calcium binding on the unliganded enzyme with either donor-acceptor pair. However, formation of the phosphoenzyme intermediate results in a measurable increase in the transfer efficiency between IAEDANS and DABmal (or Fmal); this increase is reversible upon phosphoenzyme destabilization by subsequent addition of calcium. There is no corresponding change in the intermolecular component of fluorescence resonance energy transfer between IAEDANS and fluorescein 5-isothiocyanate, indicating that the change in fluorescence resonance energy transfer probably occurs as a result of reorientation of associated ATPase polypeptide chains with respect to one another.
Animals↗
Frequency-domain fluorescence spectroscopy resolves the location of maleimide-directed spectroscopic probes within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum.
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.
Amino Acid Sequence↗
Calcium and lanthanide binding in the sarcoplasmic reticulum ATPase.
The interactions of calcium and lathanides with the sarcoplasmic reticulum ATPase, and their respective ability to activate the enzyme, were studied by direct measurements of binding with radioactive tracers, functional effects on the ATPase partial reactions, changes in the quantum yield of tryptophanyl residues and a covalently bound fluorescein label (fluorescein 5-isothiocyanate, FITC), and energy transfer between bound lanthanide and fluorescent labels. We find that: (a) Lanthanides displace calcium from specific ATPase sites with diphasic kinetics that are consistent with sequential exchange. (b) Lanthanides in excess of the calcium stoichiometry are mostly bound to sarcoplasmic reticulum lipids and non-ATPase proteins. (c) Both calcium and lanthanides activate the ATPase and allow formation of the phosphorylated intermediate by utilization of ATP; however, hydrolytic cleavage of the intermediate formed in the presence of lanthanides occurs at a slower rate than the intermediate formed in the presence of calcium. (d) In contrast to a calcium-dependent change in the quantum yield of both the tryptophanyl residues (transmembrane region) and the FITC label (extramembranous region), lanthanides induce only a change in the quantum yield of the FITC label. (e) Measurements of energy transfer between bound lanthanide and fluorescent labels detect lanthanide bound midway between the catalytic site in the globular region of the ATPase outside the membrane, and the transmembrane calcium binding domain which is involved in enzyme activation (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989a) Nature 339, 476-478). It is apparent that cation bound in this midway location controls exchange of calcium bound in the transmembrane region. The possibility that the midway location may provide a domain for binding of a second calcium is discussed.
Adenosine Triphosphatases↗