Kenneth Straub

The reaction of the psoralens with deoxyribonucleic acid

Psoralen photochemistry is specific for nucleic acids and is better understood at the molecular level than are all other methods of chemical modification of nucleic acids. These compounds are used both for in vivo structure analysis and for photochemotherapy since they easily penetrate both cells and virus particles. Apparently, natural selection has selected for membrane and virus penetrability during the evolution of these natural products. Most cells are unaffected by relatively high concentrations of psoralens in the absence of ultraviolet light, and the metabolites of the psoralens have thus far not created a problem. Finally, psoralens form both monoadduct and cross-links in nucleic acid helices, the yield of each being easily controlled by the conditions used during the photochemistry.

Structure of the unphosphorylated STAT5a dimer

STAT proteins have the function of signaling from the cell membrane into the nucleus, where they regulate gene transcription. Latent mammalian STAT proteins can form dimers in the cytoplasm even before receptor-mediated activation by specific tyrosine phosphorylation. Here we describe the 3.21-Å crystal structure of an unphosphorylated STAT5a homodimer lacking the N-terminal domain as well as the C-terminal transactivation domain. The overall structure of this fragment is very similar to phosphorylated STATs. However, important differences exist in the dimerization mode. Although the interface between phosphorylated STATs is mediated by their Src-homology 2 domains, the unphosphorylated STAT5a fragment dimerizes in a completely different manner via interactions between their β-barrel and four-helix bundle domains. The STAT4 N-terminal domain dimer can be docked onto this STAT5a core fragment dimer based on shape and charge complementarities. The separation of the dimeric arrangement, taking place upon activation and nuclear translocation of STAT5a, is demonstrated by fluorescence resonance energy transfer experiments in living cells.

The Glutamine Hydrolysis Function of Human GMP Synthetase IDENTIFICATION OF AN ESSENTIAL ACTIVE SITE CYSTEINE

GMP synthetase (EC 6.3.5.2) is an amidotransferase that catalyzes the amination of xanthosine 5′-monophosphate to form GMP in the presence of glutamine and ATP. Glutamine hydrolysis produces the necessary amino group while ATP hydrolysis drives the reaction. Ammonia can also serve as an amino group donor. GMP synthetase contains two functional domains, which are well coordinated. The “glutamine amide transfer” or glutaminase domain is responsible for glutamine hydrolysis. The synthetase domain is responsible for ATP hydrolysis and GMP formation. Inorganic pyrophosphate inhibits the synthetase and uncouples the two domain functions by allowing glutamine hydrolysis to take place in the absence of ATP hydrolysis or GMP formation. Acivicin, a glutamine analog, selectively abolishes the glutaminase activity. It inhibits the synthetase activity only when glutamine is the amino donor. When ammonia is used in place of glutamine, acivicin has no effect on the synthetase activity. Acivicin inhibits GMP synthetase irreversibly by covalent modification. Enzyme inactivation is greatly facilitated by the presence of substrates. Acivicin labels GMP synthetase at a single site, and a tryptic peptide containing the modified residue was isolated. Mass spectrometry and Edman sequence analysis show that Cys is the site of modification. This residue is conserved among GMP synthetases and is located within a predicted glutamine amide transfer domain. These data suggest that Cys is an essential residue involved in the hydrolysis of glutamine to produce an amino group and is not needed for the hydrolysis of ATP or amination of xanthosine 5′-monophosphate to produce GMP.

The methylenetetrahydrofolate-mediated biosynthesis of ribothymidine in the transfer-RNA of Streptococcus faecalis: Incorporation of hydrogen from solvent into the methyl moiety

Abstract The methyl carbon of ribothymidine in the tRNA of Streptococcus faecalis is derived from 5,10-methylenetetrahydrofolate, not S -adenosylmethionine. Isotope labeling experiments have shown that the reduction of the methylene carbon of the folate cofactor to the methyl carbon of the modified residue involves a mechanism in which hydrogen from solvent is incorporated into the methyl moiety. Although the identity of the reducing agent involved directly in this novel methylation remains to be established, data suggest that reduced flavin serves this function in vitro .

6-azidobenzo[a]pyrene. A photoactive derivative of benzo[a]pyrene for photoaffinity labeling

The synthesis, photochemical break-down and photobinding to DNA in vitro of 6-azidobenzo[a]pyrene are described.