Investigating the Interaction Between Hcf106 Peptides and the Phospholipid Bilayer by Solid-State NMR Spectroscopy

Abstract

In plant cells most thylakoid lumen proteins are synthesized in the cytosol as higher molecular weight precursors containing targeting sequences, which are imported into chloroplasts and localized to thylakoid lumen for function. Two thylakoid transport systems direct precursors to the thylakoid lumen. Our lab focuses on one of those transport systems: cpTat (chloroplast Twin arginine translocation) system, which transports fully folded proteins utilizing the trans-thylakoidal proton motive force as its only energy source. The cpTat pathway consists of three membrane-bound subunits, Tha4, Hcf106, and cpTatC. cpTatC and Hcf106 form a receptor complex where the precursor binds to initiate the translocation events, whereas Tha4 is initially found as a separate complex that joins the receptor once the precursor has bound. Hcf106 is predicted to contain a N-terminal transmembrane domain (TMD), followed by an amphipathic helix (APH) and an unstructured C-terminus. However, detailed structural information of Hcf106 remains unknown. In this study, peptides encompassing the Hcf106 TMD or APH domain were incorporated into POPC and POPC/MGDG vesicles and analyzed by solid-state NMR spectroscopy. Using 31P NMR we show that Hcf106 TMD/APH interact with the phospholipid headgroups. 2H-NMR revealed the perturbation of the deuterated acyl chain by the peptides. Lastly we used CD spectroscopy to analyze the secondary structure of Hcf106 TMD and APH peptides. The experimental results suggest: (1) Hcf106 TMD and APH peptides interact with both POPC and POPC/MGDG lipids with the lipid composition influencing the peptide-lipid interaction. (2) Hcf106 TMD and APH form alpha helical structures to varying degrees under the same conditions. (3) Hcf106 APH appears to be parallel to the membrane surface with a slight tilt into the hydrophobic region of phospholipids, whereas the TMD peptide appears to be perpendicular to the membrane surface.