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Protonation of acidic residues in the sarcoplasmic reticulum Ca2+-ATPase (SERCA 1a) was studied by multiconformation continuum electrostatic calculations in the Ca2+-bound state Ca2E1, in the Ca2+-free state E2(TG) with bound thapsigargin, and in the E2P (ADP-insensitive phosphoenzyme) analog state with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{MgF}}_{4}^{2-}\end{equation*}\end{document} \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{E}}2({\mathrm{TG}}+{\mathrm{MgF}}_{4}^{2-}).\end{equation*}\end{document} Around physiological pH, all acidic Ca2+ ligands (Glu309, Glu771, Asp800, and Glu908) were unprotonated in Ca2E1; in E2(TG) and \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{E}}2({\mathrm{TG}}+{\mathrm{MgF}}_{4}^{2-})\end{equation*}\end{document} Glu771, Asp800, and Glu908 were protonated. Glu771 and Glu908 had calculated pKa values larger than 14 in E2(TG) and \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{E}}2({\mathrm{TG}}+{\mathrm{MgF}}_{4}^{2-}),\end{equation*}\end{document} whereas Asp800 titrated with calculated pKa values near 7.5. Glu309 had very different pKa values in the Ca2+-free states: 8.4 in \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{E}}2({\mathrm{TG}}+{\mathrm{MgF}}_{4}^{2-})\end{equation*}\end{document} and 4.7 in E2(TG) because of a different local backbone conformation. This indicates that Glu309 can switch between a high and a low pKa mode, depending on the local backbone conformation. Protonated Glu309 occupied predominantly two main, very differently orientated side-chain conformations in \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{E}}2({\mathrm{TG}}+{\mathrm{MgF}}_{4}^{2-}):\end{equation*}\end{document} one oriented inward toward the other Ca2+ ligands and one oriented outward toward a protein channel that seems to be in contact with the cytoplasm. Upon deprotonation, Glu309 adopted completely the outwardly orientated side-chain conformation. The contact of Glu309 with the cytoplasm in \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{E}}2({\mathrm{TG}}+{\mathrm{MgF}}_{4}^{2-})\end{equation*}\end{document} makes this residue unlikely to bind lumenal protons. Instead it might serve as a proton shuttle between Ca2+-binding site I and the cytoplasm. Glu771, Asp800, and Glu908 are proposed to take part in proton countertransport.

A general method to study the phosphate group of phosphoenzymes with infrared difference spectroscopy by helper enzyme-induced isotope exchange was developed. This allows the selective monitoring of the phosphate P-O vibrations in large proteins, which provides detailed information on several band parameters. Here, isotopic exchange was achieved at the oxygen atoms of the catalytically important phosphate group that transiently binds to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a). [gamma-(18)O(3)]ATP phosphorylated the ATPase, which produced phosphoenzyme that was initially isotopically labeled. The helper enzyme adenylate kinase regenerated the substrate ATP from ADP (added or generated upon ATP hydrolysis) with different isotopic composition than used initially. With time this produced the unlabeled phosphoenzyme. The method was tested on the ADP-insensitive phosphoenzyme state of the Ca(2+)-ATPase for which the vibrational frequencies of the phosphate group are known, and it was established that the helper enzyme is effective in mediating the isotope exchange process.

Adenylate kinase (AdK) and apyrase were employed as helper enzymes to remove ADP in infrared spectroscopic experiments that study the sarcoplasmic reticulum Ca2+-ATPase. The infrared absorbance changes of their enzymatic reactions were characterized and used to monitor enzyme activity. AdK transforms ADP to ATP and AMP, whereas apyrase consumes ATP and ADP to generate AMP and inorganic phosphate. The benefits of using them as helper enzymes are severalfold: i), both remove ADP generated after ATP hydrolysis by ATPase, which enables repeat of ATP-release experiments several times with the same sample without interference by ADP; ii), AdK helps maintain the presence of ATP for a longer time by regenerating 50% of the initial ATP; iii), apyrase generates free Pi, which can help stabilize the ADP-insensitive phosphoenzyme (E2P); and iv), apyrase can be used to monitor ADP dissociation from transient enzyme intermediates with relatively high affinity to ADP, as shown here for ADP dissociation from the ADP-sensitive phosphoenzyme intermediate (Ca2E1P). The respective infrared spectra indicate that ADP dissociation relaxes the closed conformation immediately after phosphorylation partially back toward the open conformation of Ca2E1 but does not trigger the transition to E2P. The helper enzyme approach can be extended to study other nucleotide-dependent proteins.

Infrared spectroscopy was used to monitor the conformational change of 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) binding to the sarcoplasmic reticulum Ca(2+)-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by beta,gamma-iminoadenosine 5'-triphosphate (AMPPNP) after AMPPNP release from P(3)-1-(2-nitrophenyl)ethyl AMPPNP (caged AMPPNP). The resulting infrared difference spectra are compared to those of AMPPNP binding to the free ATPase, to obtain a difference spectrum that reflects solely TNP-AMP binding to the Ca(2+)-ATPase. TNP-AMP used as an ATP analog in the crystal structure of the sarcoplasmic reticulum Ca(2+)-ATPase was found to induce a conformational change upon binding to the ATPase. It binds with a binding mode that is different from that of AMPPNP, ATP, and other tri- and diphosphate nucleotides: TNP-AMP binding causes partially opposite and smaller conformational changes compared to ATP or AMPPNP. The conformation of the TNP-AMP ATPase complex is more similar to that of the E1Ca(2) state than to that of the E1ATPCa(2) state. Regarding the use of infrared spectroscopy as a technique for ligand binding studies, our results show that infrared spectroscopy is able to distinguish different binding modes.

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding were recorded in H(2)O and (2)H(2)O at -7 degrees C and pH 7.0. The reaction cycle was triggered by the photochemical release of nucleotides (ATP, ADP, and AMP-PNP) from a biologically inactive precursor (caged ATP, P(3)-1-(2-nitrophenyl) adenosine 5'-triphosphate, and related caged compounds). Infrared absorbance changes due to ATP release and two steps of the Ca(2+)-ATPase reaction cycle, ATP binding and phosphorylation, were followed in real time. Under the conditions used in our experiments, the rate of ATP binding was limited by the rate of ATP release (k(app) congruent with 3 s(-1) in H(2)O and k(app) congruent with 7 s(-1) in (2)H(2)O). Bands in the amide I and II regions of the infrared spectrum show that the conformation of the Ca(2+)-ATPase changes upon nucleotide binding. The observation of bands in the amide I region can be assigned to perturbations of alpha-helical and beta-sheet structures. According to similar band profiles in the nucleotide binding spectra, ATP, AMP-PNP, and ADP induce similar conformational changes. However, subtle differences between ATP and AMP-PNP are observed; these are most likely due to the protonation state of the gamma-phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the gamma-phosphate group in the interactions between the Ca(2+)-ATPase and the nucleotide. Nucleotide binding affects Asp or Glu residues, and bands characteristic of their protonated side chains are observed at 1716 cm(-1) (H(2)O) and 1706 cm(-1) ((2)H(2)O) and seem to depend on the charge of the phosphate groups. Bands at 1516 cm(-1) (H(2)O) and 1514 cm(-1) ((2)H(2)O) are tentatively assigned to a protonated Tyr residue affected by nucleotide binding. Possible changes in Arg, Trp, and Lys absorption and in the nucleoside are discussed. The spectra are compared with those of nucleotide binding to arginine kinase, creatine kinase, and H-ras P21.

Phosphate binding to the sarcoplasmic reticulum Ca2+-ATPase was studied by time-resolved Fourier transform infrared spectroscopy with ATP and isotopically labeled ATP ([beta-18O2, betagamma-18O]ATP and [gamma-18O3]ATP). Isotopic substitution identified several bands that can be assigned to phosphate groups of bound ATP: bands at 1260, 1207, 1145, 1110, and 1085 cm(-1) are affected by labeling of the beta-phosphate, bands likely near 1154, and 1098-1089 cm(-1) are affected by gamma-phosphate labeling. The findings indicate that the strength of interactions of beta- and gamma- phosphate with the protein are similar to those in aqueous solution. Two bands, at 1175 and 1113 cm(-1), were identified for the phosphate group of the ADP-sensitive phosphoenzyme Ca2E1P. They indicate terminal and bridging P-O bond strengths that are intermediate between those of ADP-insensitive phosphoenzyme E2P and the model compound acetyl phosphate in water. The bridging bond of Ca2E1P is weaker than for acetyl phosphate, which will facilitate phosphate transfer to ADP, but is stronger than for E2P, which will make the Ca2E1P phosphate less susceptible to attack by water.

P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP (pHP-caged ATP) has been investigated for its application as a phototrigger for the rapid activation of electrogenic ion pumps. The yield of ATP after irradiation with a XeCl excimer laser (lambda = 308 nm) was determined at pH 6.0-7.5. For comparison, the photolytic yields of P(3)-[1-(2-nitrophenyl)]ethyl ATP (NPE-caged ATP) and P(3)-[1, 2-diphenyl-2-oxo]ethyl ATP (desyl-caged ATP) were also measured. It was shown that at lambda = 308 nm pHP-caged ATP is superior to the other caged ATP derivatives investigated in terms of yield of ATP after irradiation. Using time-resolved single-wavelength IR spectroscopy, we determined a lower limit of 10(6) s(-1) for the rate constant of release of ATP from pHP-caged ATP at pH 7.0. Like NPE-caged ATP, pHP-caged ATP and desyl-caged ATP bind to the Na(+), K(+)-ATPase and act as competitive inhibitors of ATPase function. Using pHP-caged ATP, we investigated the charge translocation kinetics of the Na(+),K(+)-ATPase at pH 6.2-7.4. The kinetic parameters obtained from the electrical measurements are compared to those obtained with a technique that does not require caged ATP, namely parallel stopped-flow experiments using the voltage-sensitive dye RH421. It is shown that the two techniques yield identical results, provided the inhibitory properties of the caged compound are taken into account. Our results demonstrate that under physiological (pH 7.0) and slightly basic (pH 7.5) or acidic (pH 6. 0) conditions, pHP-caged ATP is a rapid, effective, and biocompatible phototrigger for ATP-driven biological systems.

Several biophysical techniques have been used to determine differences in the aggregation profile (i.e., the secondary structure, aggregation propensity, dynamics, and morphology of amyloid structures) and the effects on cell viability of three variants of the amyloid beta peptide involved in Alzheimer's disease. We focused our study on the Glu22 residue, comparing the effects of freshly prepared samples and samples aged for at least 20 days. In the aged samples, a high propensity for aggregation and beta-sheet secondary structure appears when residue 22 is capable of establishing polar (Glu22 in wild-type) or hydrophobic (Val22 in E22V) interactions. The Arctic variant (E22G) presents a mixture of mostly disordered and alpha-helix structures (with low beta-sheet contribution). Analysis of transmission electron micrographs and atomic force microscopy images of the peptide variants after aging showed significant quantitative and qualitative differences in the morphology of the formed aggregates. The effect on human neuroblastoma cells of these Abeta(12-28) variants does not correlate with the amount of beta-sheet of the aggregates. In samples allowed to age, the native sequence was found to have an insignificant effect on cell viability, whereas the Arctic variant (E22G), the E22V variant, and the slightly-aggregating control (F19G-F20G) had more prominent effects.

Protonation of the Ca2+ ligands of the SR Ca2+-ATPase (SERCA1a) was studied by a combination of rapid scan FTIR spectroscopy and electrostatic calculations. With FTIR spectroscopy, we investigated the pH dependence of C=O bands of the Ca2+-free phosphoenzyme (E2P) and obtained direct experimental evidence for the protonation of carboxyl groups upon Ca2+ release. At least three of the infrared signals from protonated carboxyl groups of E2P are pH dependent with pKa values near 8.3: a band at 1758 cm−1 characteristic of nonhydrogen-bonded carbonyl groups, a shoulder at 1720 cm−1, and part of a band at 1710 cm−1, both characteristic of hydrogen-bonded carbonyl groups. The bands are thus assigned to H+ binding residues, some of which are involved in H+ countertransport. At pH 9, bands at 1743 and 1710 cm−1 remain which we do not attribute to Ca2+/H+ exchange. We also obtained evidence for a pH-dependent conformational change in β-sheet or turn structures of the ATPase. With MCCE on the E2P analog E2(\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{TG}}+{\mathrm{MgF}}_{4}^{2-}\end{equation*}\end{document}), we assigned infrared bands to specific residues and analyzed whether or not the carbonyl groups of the acidic Ca2+ ligands are hydrogen bonded. The carbonyl groups of Glu771, Asp800, and Glu908 were found to be hydrogen bonded and will thus contribute to the lower wave number bands. The carbonyl group of some side-chain conformations of Asp800 is left without a hydrogen-bonding partner; they will therefore contribute to the higher wave number band.

Sodium dodecyl sulfate has been proven as an amyloid-like misfolding agent [1-3]. Our study comprises the biophysical characterization of human peptides in the presence of submicellar and micellar concentrations of SDS. The prodynorphin derived peptides (Big dynorphin, dynorphin A and dynorphin B) [4], the amyloid β peptide [5], and the proinsulin derived C-peptide are our subject of study. As determined by CD and FTIR spectroscopy, the peptide structural transitions involve different secondary structures, such as random coil, β-sheet and α-helix. By means of NMR, dynamic light scattering, native-PAGE or ThT fluorescence, we have shown that all the peptides transit through a high molecular weight aggregated state at submicellar detergent concentrations. Finally, studies with model membranes with different charge composition have been carried out to relate the structural characterization of these peptides to their possible role in the cell and their action mechanisms in pathology.1. Rangachari, V., Moore, B.D., Reed, D.K., Sonoda, L.K., Bridges, A.W., Conboy, E., Hartigan, D., and Rosenberry, T.L. (2007). Biochemistry 46, 12451-12462.2. Rangachari, V., Reed, D.K., Moore, B.D., and Rosenberry, T.L. (2006). Biochemistry 45, 8639-8648.3. Tew, D.J., Bottomley, S.P., Smith, D.P., Ciccotosto, G.D., Babon, J., Hinds, M.G., Masters, C.L., Cappai, R., and Barnham, K.J. (2008). Biophys J 94, 2752-2766.4. Hugonin, L., Barth, A., Graslund, A., and Peralvarez-Marin, A. (2008). Biochim Biophys Acta. in press.5. Wahlstrom, A., Hugonin, L., Peralvarez-Marin, A., Jarvet, J., and Graslund, A. (2008). FEBS J. in press.