Chloroplasts are organelles responsible for photosynthesis in plants. They are bounded by a double membrane envelope and contain an internal membrane system (the site of photosynthesis), the thylakoid bounding a small aqueous lumen. All of the proteins found in the thylakoid lumen of chloroplasts are encoded by nuclear genes, synthesized in the cytoplasm, and must cross the three membrane systems <i>en route</i> to their final destination. The chloroplast Twin Arginine Translocation (cpTat) system facilitates transport across the thylakoid of about half of the lumen proteins. Biochemical research has revealed considerable information about the transport of proteins into the thylakoid lumen by cpTat. Proteins transported into the lumen are vital for the proper functioning of the thylakoid in photosynthesis. However, experimental evidence regarding the structure of cpTat proteins in thylakoid membranes is lacking. In particular, we are interested in the topology and conformational changes of one of the transmembrane proteins of the cpTat system, Hcf106, in the presence or absence of the precursor substrate. Here, Hcf106 wild type and cysteine variant proteins were over-expressed, isolated and purified. The purified proteins were then labeled with MTSL ((1-Oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) methanethiosulfonate) and inserted into isolated thylakoids of the cyanobacterium <i>Synechocystis, </i>spp. 6803 and used for electron paramagnetic resonance spectroscopy (EPR) to report on the environment of the spin label. Such studies of the structure and topology of Hcf106 and other cpTat proteins will enable us to answer major questions of how the translocation component membrane proteins change during the translocation of precursor proteins via the cpTat pathway.

Topology and conformational changes of the chloroplast twin arginine protein, HCF106, as revealed by site-directed spin label and electron paramagnetic resonance spectroscopy

251st National Meeting (2016)

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