1 Chemical approach for evaluating role of the cysteine residues in pentameric phospholamban structure: Effect on sarcoplasmic reticulum Ca 2+ -ATPase Christine B. Karim, 1 Laxma G. Reddy, 1 Gregory W. Hunter, 1 Yvonne M. Angell, 2 George Barany, 2 and David D. Thomas 1 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455, U.S.A. and 2 Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, U.S.A. Introduction Calcium transport into the sarcoplasmic reticulum of cardiac muscle is catalyzed by the Ca pump and regulated by phospholamban (PLB), a 52-amino acid integral membrane peptide that inhibits the pump [1]. PLB is predominantly a homopentamer on electrophoresis gels (SDS-PAGE), with only a small fraction of monomer [2]. Specific residues that influence the pentameric structure of PLB are located in the hydrophobic transmembrane domain [3]. It has been previously suggested that the pentamer is stabilized by interhelical interactions between leucines (residues 37, 44, and 51) and isoleucines (residues 40 and 47), forming a leucine/isoleucine zipper [3]. The function of the three Cys residues (36, 41, and 46) within this domain is not known. It is assumed from Cys mutagenesis experiments that Cys residues are essential for stable pentamers [4], although they are not involved in disulfide bonding [2]. The function of the N- terminal acetyl group (Fig. 1) is also not known, although its removal results in a greater positive charge on the cytoplasmic domain of the peptide under normal biological conditions. We have designed and synthesized a PLB derivative containing no cysteines, and a PLB derivative lacking an N-terminal acetyl cap, to evaluate the contribution of the transmembrane cysteines and the N-terminal acetyl group in the function and pentameric structure of PLB, respectively. Results and Discussion To determine the roles of the three Cys residues in the transmembrane domain of PLB, we have used Fmoc solid-phase peptide synthesis to prepare PLB derivatives in which each of the three cysteines are replaced with alanines (Ala-PLB). Acetyl-MDKVQYLTRSAIRRASTIEMPQQARQNLQNLFINF CLILICLLLICIIVMLL 50 40 30 20 10 1 Cytoplasmic Transmembrane Figure 1: Amino acid sequence of phospholamban.