The chloroplast Twin Arginine Translocase (cpTAT) system transports fully folded proteins across the thylakoid membrane in plant cells using only energy derived from the proton motive force (PMF). Three membrane bound component proteins: cpTatC, Hcf106, and Tha4 function together in a transient manner to accomplish transport. However, clear mechanistic details of this process remain elusive such as how cpTAT utilizes energy stored in the PMF or how the individual component proteins interact during each step of transport. In addition, prior structural characterization of (cp)TAT proteins used truncated versions of the components. This dissertation describes work to develop methods to purify full-length Hcf106 for biophysical characterization. Additionally, this dissertation details the work to determine the function of a membrane embedded glutamate in the Tha4 transmembrane helix (TMH).A series of purification trials were carried out to isolate Hcf106 fused to maltose binding protein (MBP) by the recognition sequence of tobacco etch virus protease (TEVp). Fusion protein and protease were expressed in and purified from E. coli using affinity chromatography. Multiple parameters and additives were tested during optimization of TEVp proteolysis reactions with MBP-Hcf106. TEVp and free MBP were separated from un-cleaved MBP-Hcf106 and free Hcf106 by affinity and size exclusion chromatography. Although TEVp and free MBP were removed after an optimized proteolysis reaction, free Hcf106 showed its recalcitrant nature through resistance of separation from un-cleaved MBP-Hcf106 by size exclusion chromatography in several detergent and buffer conditions.To better understand the role of the membrane embedded Tha4 glutamate 10 (E10), Tha4 variants with glutamate to alanine (E10A) or glutamate to aspartate (E10D) substitutions were used to complement loss of cpTAT function in thylakoid membranes. Sequential glutamate substitutions in the TMH of Tha4 variant E10A were unable to restore transport while aspartate substitutions were mildly able to complement loss of function. Furthermore, organization between three structural regions in Tha4 E10/A/D variants was determined by disulfide crosslinking during various transport conditions. Tha4 E10/A/D variant oligomer formation was enhanced in the presence of functional precursor with and without PMF present. An increase in TMH hydrophobicity by alanine substitution was shown to increase Tha4 stability in isolated thylakoid membranes and to promote tighter packing interactions between adjacent Tha4 monomers. The interaction data was then used to develop a model of how Tha4 E10/A/D variant tetramers pack and reorganize in the presence of precursor. Advisors/Committee Members: Dabney-Smith, Carole (Advisor), Page, Rick (Committee Chair).