This project focuses on the structure--function relationships, determination of amino acid sequences and identification and characterization of post-translational modifications of proteins. In continuing studies a new class of anti--HIV plant proteins, MAP 30, MAP 30II TAP 29, GAP 31, DAP 32 and 30, have been identified and characterized. These single chain ribosome--inactivating proteins inhibit HIV--1 infection as well as replication, as measured by syncytium formation, viral core protein p24 expression, and HIV--associated reverse transcriptase. Furthermore, these proteins all exhibit topoisomerase-- like activity and undetectable cytotoxicity in host cells or insignificant toxicity in mice. Molecular weights of several of these proteins were determined by electrospray ionization mass spectroscopy. All were shown to be smaller than those determined by SDS--PAGE: MAP 30=29106, MAP 30II=28587, TAP 29=27168 and GAP 31=29187. Crystallization of GAP 31 was achieved in 1.6 M ammonium sulfate and 0.05 M Tris, pH 8.5 at 28 degrees C after two weeks. The crystal diffracts up to 2.0 angstroms resolution and belongs to monoclinic space group ?2/1. The cell dimensions are a=49.30(2) angstroms, b=44.57(2) angstroms, c=137.78(7) angstroms and beta=98.32(3) degrees. The crystal contains two molecules of GAP 31 per asymmetric unit and 49% solvent. A detailed crystallographic study of GAP 31 by molecular replacement method using known ricin coordinates as a copy is in progress. The identification of protein kinase C phosphorylation sites in MARCKS, neurogranin, and neuromodulin allows the elucidation of dephosphorylation specificities of calcineurin and protein phosphatases 1 and 2A. Co--localization of these phosphatases and PKC substrates and high concentration of calcineurin are consistent with the notion of interacting--regulatory actions among protein phosphatases, protein kinases, and calmodulin. The amino acid sequence analysis of fragments upon proteolysis of a number of prohormone peptides by yeast aspartic protease 3 (YAP3) permitted determination of specificity of the protease. YAP3 cleaved proinsulin at the paired--basic amino acid residue site of the B--C chain junction as well as the C--A chain junction. This protease generated Leu--enkephalin and Leu--enkephalin--Arg from dynorphin A (1-11) and dynorphin B (1--13) respectively, indicating cleavages at a paired-- basic residue site motif which has an Arg or Lys at the downstream P4' position, respectively. Porcine cholecystokinin 33 was cleaved at two mono--basic amino acid residue sites at Lys/23 and Arg/9 which contain an upstream Arg at the P6 and P5 position, respectively. These results show that YAP3 recognizes three different motifs: paired--basis amino acid residues sites containing an additional basic residue at the upstream P4-- P6 or downstream P4'--P6' positions.
