The twin-arginine translocation (Tat(1)) pathway is unique with respect to its property to translocate proteins in a fully folded conformation across ion-tight membranes. In chloroplasts and Gram-negative bacteria, Tat translocase consists of the integral subunits TatB and TatC, which are assumed to constitute the membrane receptor, and TatA, a bitopic membrane protein being responsible in a yet unknown manner for the membrane translocation step. Antibody inhibition of intrinsic thylakoidal TatA activity and recovery of transport by heterologously expressed, purified TatA allowed to exactly quantify the amount of TatA required to catalyse membrane transport of the model Tat substrate 16/23. We can show that TatA concentrations in the 100nM range are sufficient to efficiently catalyse membrane transport of the protein, which corresponds well to the amount of TatA identified in thylakoids. Furthermore, TatA shows cooperativity in its catalytic activity suggesting that Tat translocase operates as an allosteric enzyme complex.

Significant photoswitching ability is found for secondary thioxopeptide bonds and can be used for the photomodulation of the backbone conformation of peptides or proteins.

No abstract

Oligopeptides derived from the gag polyprotein (Pr55 gag) of human immunodeficiency virus type 1 (HIV-1) segment were used to evaluate the extension of the putative binding region for the complex of Pr55 gag and the human cytosolic peptidyl prolyl cis/trans isomerase (PPIase) 18 kDa cyclophilin (Cypl8). Five N-terminally acetylated, C-terminally amidated oligopeptides containing one (HIV-1 Gag218-224; 1), two (HIV-1 Gag 21s-226 and HIV-1 Gag217-224; 2 and 3, respectively), three (HIV-I Gag217-226; 4) or four (HIV-1 Gag213-237; 5) proline residues were synthesized. Using competition experiments with a standard substrate the binding affinities to Cypl8 of the synthesized peptides were determined. The ICs0 value of 184 ~M for the 25-mer peptide 5 was fivefold or more lower than those of the peptides 1--4 lacking one or more prolines. Failure of competition in assays containing enzymes of other PPlase families by millimolar concentrations of 5 revealed a Cyp18 specific interaction involving the active site of the enzyme. In its far UV circular dichroism, aqueous solutions of 5 display properties of random coil conformation, but spectra were also consistent with a small contribution of proline specific secondary structures. However, a proline-rich peptide typical of forming left-handed polyproline II helices did not compete for the active site of Cypl8. The results demonstrate that the putative binding region of HIV-1 gag polyprotein has a certain degree of binding affinity to the PPIase site of Cypl8, and may add a previously unrecognized topological component to the known subsite specificity of cyciophilins.

In chloroplasts and bacteria, the Tat (twin-arginine translocation) system is engaged in transporting folded passenger proteins across the thylakoid and cytoplasmic membranes, respectively. To date, three membrane proteins (TatA, TatB, and TatC) have been identified to be essential for Tat-dependent protein translocation in the plant system, whereas soluble factors seem not to be required. In contrast, in the bacterial system, several cytosolic chaperones were described to be involved in Tat transport processes. Therefore, we have examined whether stromal or peripherally associated membrane proteins also play a role in Tat transport across the thylakoid membrane. Analyzing both authentic precursors as well as the chimeric 16/23 protein, which allows us to study each step of the translocation process individually, we demonstrate that a soluble form of TatA is present in the chloroplast stroma, which significantly improves the efficiency of Tat-dependent protein transport. Furthermore, this soluble TatA is able to reconstitute the Tat transport properties of thylakoid membranes that are transportincompetent due to extraction with solutions of chaotropic salts.