| ATP synthase is the key enzyme in energy conversion on transducing membranes such as chloroplast thylakoid membranes, mitochondrial inner membranes and bacterial plasma membranes. The general structural features of the enzyme are highly conserved among different organisms. In chloroplast, the enzyme consists of two parts, CFo and CF1. CFo, a membrane-spanning complex, conducts proton flux through the thylakoid membrane and provides an affinity binding site for the CF1 complex. CF1 is extrinsic to the membrane, contains nucleotide-binding and catalytic sites, can hydrolyze ATP at high rates after appropriate treatment, and consists of five types of subunits in a stoichiometry of (????(???The area I focused on most is structure and function study of the smaller subunits (subunits????(?and??) of CF1 part of spinach chloroplast ATP synthase. 1. The conserved residue Thr42 of ?-subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded??-proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca2+-ATPase activity and blocking proton gate) as the native ε subunit isolated from chloroplasts. The??T42C and??T42R showed higher inhibitory activities on the soluble CF1(–?) Ca2+-ATPase than the εWT. The εT42I inhibited the Ca2+-ATPase activity of soluble CF1 and restored photophosphorylation activity of membrane-bound CF1 deficient in ??the most efficiently. Far-ultraviolet CD spectra showed that the portions of α-helix and β-sheet structures of the three mutants were somewhat different from ?WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the??-subunit and the basic functions of the??-subunit as an inhibitor of Ca2+-ATPase and the proton gate are related.2. Mutagenesis was used to generate seven truncation mutants of the spinach (Spinacia oleracea) chloroplast ATP synthase ( subunit lacking 5, 11, 17, or 35 amino acid residues from the N-terminus or 3, 9, or 15 residues from the C-terminus. Interactions between these mutants and all other subunits of the chloroplast ATPase were investigated by a yeast two-hybrid system. The results indicate that the N-terminal deletions mainly affected interactions between the ( subunit and the other part of CF1, but did not significantly affect interactions with the CFo sector. In contrast, C-terminal truncations of the ( subunit mainly affected its interaction with the CFo sector and caused little impairment in interactions with the other part of CF1. The conformation of the ( subunit C-terminal domain seems to be more sensitive to the truncations, as shown by minimal expression driven by C-terminal deleted (nine residues) mutants. Further studies showed C-terminal truncations of the ( subunit greatly impaired its ability to restore cyclic photophosphorylation in NaBr vesicles, whereas N-terminal truncations had little effect on the ability of ( to plug the CFo channel. None of the mutants impaired ATP hydrolysis by CF1-ATPase.3. Five truncation mutants of chloroplast ATP synthase???subunit from spinach (Spinacia oleracea) lacking 8, 12, 16, 20 or 60 amino acid residues from the N-terminus, were generated by PCR by a mutagenesis method. The recombinant???genes were overexpressed in E. coli and assembled with a native (( complex. The?((?WT assembly exhibited high Mg2+- or Ca2+-ATPase activities. Deletions of 8 residues of the???subunit N-terminus caused decrease in rates of ATP hydrolysis to 30% of that of ((?WT assembly. Furthermore, only ~6% of rates of ATP hydrolysis was retained with the sequential deletions of????subunit up to 20 residues compared with the activities of ((?WT assembly. The inhibitory effect of the???subunit on ATP hydrolysis of those ((??assemblies varied to a large extent. These observations indicate that the ??subunit N-terminal end is very important, together with other regions of the???subunit, in st...
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