Investigating Structural Changes in the Lipid Bilayer upon Insertion of the Transmembrane Domain of the Membrane-Bound Protein Phospholamban Utilizing 31P and 2H Solid-State NMR Spectroscopy

Abstract

Phospholamban (PLB) is a 52-amino acid integral membrane protein that regulates the flow of Ca 2+ ions in cardiac muscle cells. In the present study, the transmembrane domain of PLB (24–52) was incorporated into phospholipid bilayers prepared from 1-palmitoyl-2-oleoyl- sn-glycero-phosphocholine (POPC). Solid-state 31P and 2H NMR experiments were carried out to study the behavior of POPC bilayers in the presence of the hydrophobic peptide PLB at temperatures ranging from 30°C to 60°C. The PLB peptide concentration varied from 0 mol % to 6 mol % with respect to POPC. Solid-state 31P NMR spectroscopy is a valuable technique to study the different phases formed by phospholipid membranes. 31P NMR results suggest that the transmembrane protein phospholamban is incorporated successfully into the bilayer and the effects are observed in the lipid lamellar phase. Simulations of the 31P NMR spectra were carried out to reveal the formation of different vesicle sizes upon PLB insertion. The bilayer vesicles fragmented into smaller sizes by increasing the concentration of PLB with respect to POPC. Finally, molecular order parameters ( S CD) were calculated by performing 2H solid-state NMR studies on deuterated POPC ( sn-1 chain) phospholipid bilayers when the PLB peptide was inserted into the membrane.