|Keywords:||Biochemistry; Physical Chemistry; Chloroplast twin arginine transport protein; Spontaneous insertion; Synthetic peptide; Multilamellar vesicles|
|Full text PDF:||http://rave.ohiolink.edu/etdc/view?acc_num=miami1429539744|
The chloroplast twin arginine transport (cpTat) system is one of the two main transport systems that direct thylakoidal proteins to the thylakoid lumen after entering the chloroplast. The cpTat system transports fully folded proteins utilizing the trans-thylakoidal proton motive force as its only energy source. It consists of three membrane-bound subunits, Tha4, Hcf106 and cpTatC. Hcf106, composes the receptor complex along with cpTatC, is the least structurally studied one among the three proteins. Hcf106 is predicted to contain a single N-terminal transmembrane domain (TMD) followed by a Pro-Gly hinge, an amphipathic a-helix (APH), and a loosely structured C terminus. However, detailed topology information of Hcf106 remains unknown. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes; however how this process occurs is not understood. In this dissertation, the biophysical technique of solid-state NMR spectroscopy and the biochemical method of cysteine accessibility labeling techniques were utilized to study the peptide-lipid interaction and the topology of Hcf106.To investigate how Hcf106 inserts itself into the membrane unassisted, solid-state NMR spectroscopy was used to investigate the membrane activity of the TMD and APH of Hcf106 separately. Peptides encompassing TMD or APH were incorporated into multilamellar vesicles (MLVs) to probe peptide-lipid interactions. Solid-state 31P NMR and 2H NMR spectroscopic experiments revealed that both peptides perturbed the headgroup and acyl chain regions of phospholipids as indicated by changes in spectral lineshape, chemical shift anisotropy width, and 2H order SCD parameters. Furthermore, the comparison between POPC (1-palmitoyl-2-oleoyl snglycero-phosphoatidylcholine) and POPC/MGDG (monogalactosyl diacylglycerol) MLVs indicated that the lipid bilayer composition affected the peptide-lipid interaction with the peptide interacting preferentially with vesicles that more closely mimic the thylakoid.The topology of Hcf106 in native thylakoid membrane was studied by cysteine accessibility labeling techniques in the absence and presence of precursor. In the resting state, Hcf106 displayed an overall lumenal N terminus and stromal C terminus with a shortened TMD and a tilted APH whose N-proximal end is more membrane integrated while the C-proximal end is more stroma accessible. During precursor binding, the overall topology of Hcf106 remains unchanged, while the N-proximal APH becomes more accessible to the stroma labeling reagent. We interpreted these data to be the result of a Hcf106 topology change such that the APH lies more parallel to the membrane normal upon precursor binding, potentially contributing to a weakening of the thylakoid membrane. We are in favor of the membrane weakening model in which a localized weakening and disruption of the thylakoid membrane around the precursor is the essential motivation for translocation.