CONFORMATION OF THE CYTOPLASMIC DOMAIN OF PHOSPHOLAMBAN BY NMR AND CD

Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectrosc opy have been used to characterize the conformation of the putative cy toplasmic domain of phospholamban (PLB), an oligomeric membrane-bound protein which regulates the activity of the cardiac sarcoplasmic retic ulum Ca2+-dependent ATPase. In aqueous solution the 25-residue peptide adopts a number of rapidly interconverting conformers with no seconda ry structural type obviously predominating. However, in trifluoroethan ol (TFE) the conformation, while still highly dynamic, is characterize d by a high proportion of helical structures. Evidence for this is pro vided by alpha CH chemical shifts and low NH chemical shift temperatur e coefficients, small NH-alpha CH intraresidue scalar coupling constan ts, a substantial number of distinctive interresidue nuclear Overhause r effects (NOEs) [d(NN)(i, i+1), d(alpha N)(i, i+3), d(alpha beta)(i, i+3) and d(alpha N)(i, i+4)] and characteristic CD bands at 190 (posit ive), 206 (negative) and 222 nm (negative). The helicity is interrupte d around Pro-21. The activity of PLB is regulated by phosphorylation a t either Ser-16 or Thr-17. CD shows that phosphorylation at Ser-18 by the cAMP-activated protein kinase causes about an 11% decrease in alph a-helical content in TFE.