This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.107.114033v1 94/2/600    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Andersson, J. Articles by Barth, A. Search for Related Content PubMed PubMed Citation Articles by Andersson, J. Articles by Barth, A.

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

Julia Andersson ^*, Karin Hauser , Eeva-Liisa Karjalainen ^* and Andreas Barth ^*

^* Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden; and Institut für Biophysik, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany

Correspondence: Address reprint requests to Andreas Barth, Dept. of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, SE-106 91 Stockholm, Sweden. E-mail: Andreas.Barth{at}dbb.su.se.

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 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 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(), 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.