(15)N Solid-state NMR spectroscopic studies on phospholamban at its phosphorylated form at ser-16 in aligned phospholipid bilayers

Abstract

Wild-type phospholamban (WT-PLB) is a pentameric transmembrane protein that regulates the cardiac cycle (contraction and relaxation). From a physiological prospective, unphosphorylated WT-PLB inhibits sarcoplasmic reticulum ATPase activity; whereas, its phosphorylated form relieves the inhibition in a mechanism that is not completely understood. In this study, site-specifically (15)N-Ala-11- and (15)N-Leu-7-labeled WT-PLB and the corresponding phosphorylated forms (P-PLB) were incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine/2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPC/DOPE) mechanically oriented lipid bilayers. The aligned (15)N-labeled Ala-11 and Leu-7 WT-PLB samples show (15)N resonance peaks at approximately 71ppm and 75ppm, respectively, while the corresponding phosphorylated forms P-PLB show (15)N peaks at 92ppm and 99ppm, respectively. These (15)N chemical shift changes upon phosphorylation are significant and in agreement with previous reports, which indicate that phosphorylation of WT-PLB at Ser-16 alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers.

Figure 1

SDS-PAGE gels of WT-PLB and P-PLB. From left to right: P-PLB, WT-PLB, and the molecular weight marker.

Figure 2

³¹P solid-state NMR spectra of aligned DOPC/DOPE (4:1 ratio) bilayers in presence of 0 mol% (A), 1 mol% of ¹⁵N-uniformly labeled PLB (B), ¹⁵N-Ala-11 labeled WT-PLB (C) and P-PLB (E), ¹⁵N-Leu-7 labeled WT-PLB (D) and P-PLB (F), respectively.

Figure 3

¹⁵N cross-polarization NMR spectra of uniformly ¹⁵N-labeled WT-PLB (A) as well as site-specifically ¹⁵N-labeled WT-PLB at Ala-11 (B) or Leu-7 (D), and P-PLB at Ala-11 (C) or Leu-7 (E) in oriented DOPC/DOPE (4:1 ratio) lipid bilayers. The ¹⁵N NMR powder spectra of WT-PLB (F) and P-PLB (G) in POPC MLVs are also presented.