Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban

Phospholamban is a 52-amine acid residue membrane protein that regulates Ca²⁺-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca²⁺- selective ion channel. We have used polarized attenuated total reflection- Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLS. The frequency and dichroism of the amide I and II bands appearing at 1658 cm^-1 and 1544 cm^{- 1}, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidylcholine bilayer membranes is predominantly α- helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating ¹³C at two positions in the protein backbone. A small band seen near 1614 cm^-1 is assigned to the amide I mode of the ¹³C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are α-helical, with an axial orientation that is approximately 30° relative to the membrane normal. Upon exposure to ²H₂O (D₂O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope- labeled hPLB films, our results strongly support models of PLB that predict the existence of an α-helical hydrophobic region spanning the membrane domain.