PMID:17890386
| Citation |
Andersson, J, Hauser, K, Karjalainen, EL and Barth, A (2008) Protonation and hydrogen bonding of Ca2+ site residues in the E2P phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase studied by a combination of infrared spectroscopy and electrostatic calculations. Biophys. J. 94:600-11 |
|---|---|
| Abstract |
Protonation of the Ca(2+) ligands of the SR Ca(2+)-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 Ca(2+)-free phosphoenzyme (E2P) and obtained direct experimental evidence for the protonation of carboxyl groups upon Ca(2+) release. At least three of the infrared signals from protonated carboxyl groups of E2P are pH dependent with pK(a) 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 Ca(2+)/H(+) exchange. We also obtained evidence for a pH-dependent conformational change in beta-sheet or turn structures of the ATPase. With MCCE on the E2P analog E2(TG+MgF(4)(2-)), we assigned infrared bands to specific residues and analyzed whether or not the carbonyl groups of the acidic Ca(2+) ligands are hydrogen bonded. The carbonyl groups of Glu(771), Asp(800), and Glu(908) 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 Asp(800) is left without a hydrogen-bonding partner; they will therefore contribute to the higher wave number band. |
| Links |
PubMed PMC2157260 Online version:10.1529/biophysj.107.114033 |
| Keywords |
Animals; Aspartic Acid; Calcium/metabolism; Deuterium; Glutamic Acid; Hydrogen Bonding; Hydrogen-Ion Concentration; Ligands; Protein Conformation; Protons; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry; Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism; Spectrophotometry, Infrared; Static Electricity; Water |
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