The Arc repressor of bacteriophage P22 is a DNA binding protein that does not belong to any of the known classes of such proteins. We have undertaken a 1H NMR study of the protein with the aim of elucidating its three-dimensional structure in solution and its mode of binding of operator DNA. Here we present the 1H nuclear magnetic resonance (NMR) assignments of all backbone protons and most of the side-chain protons of Arc repressor. Elements of secondary structure have been identified on the basis of networks of characteristic sequential and medium-range nuclear Overhauser enhancements (NOEs). Two alpha-helical regions have been found in the peptide regions 16-29 and 35-45. The ends of the helices could not yet be firmly established and could extend to residue 31 for the first helix and to residue 49 for the second. Immediately before the first helix, between residues 8 and 14, a region is present with beta-sheet characteristics dominated by a close proximity of the alpha-protons of residues 9 and 13. Because of the dimeric nature of the protein there are still two possible ways in which the NOEs in the beta-sheet region can be interpreted. If the NOEs are intramonomer, this requires a tight turn involving residues 10-12. Alternatively, if the NOEs are intermonomer, then and antiparallel beta-sheet would be implicated comprising two strands of different Arc monomers. While the data presently do not allow an unambiguous choice between these two possibilities, some evidence is discussed that favors the latter (beta-sheet between monomers).(ABSTRACT TRUNCATED AT 250 WORDS)

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15 Nlabeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerolmethanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH 4 Cl/glucose-glycerolmethanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man 8-15 GlcNAc 2 ). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Man up-to-30 GlcNAc 2 ). The latter finding made the X-33 strain not very suitable for generating N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

The isolation of mixtures of 2-, 3-, and 4-deuteriopyridine, 2-, 3-, and 4-trimethylsilylpyridine, or 2-, 3-, and 4-methylthiopyridine indicates successful metallation of pyridine with a 1 : 1 mixture of BuLi-ButOK in tetrahydrofuran-hexane at -100°C.

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The structure and dynamics of the chymotryptic tetramerization domain of the Mnt repressor of Salmonella bacteriophage P22 have been studied by NMR spectroscopy. Two sets of resonances (A and B) were found, representing the asymmetry within the homotetramer. Triple-resonance techniques were used to obtain unambiguous assignments of the A and B resonances. Intra-monomeric NOEs, which were distinguished from the inter-monomeric NOEs by exploiting (13)C/(15)N-filtered NOE experiments, demonstrated a continuous alpha-helix of approximately seven turns for both the A and B monomers. The asymmetry facilitated the interpretation of inter-subunit NOEs, whereas the antiparallel alignment of the subunits allowed further discrimination of inter-monomeric NOEs. The three-dimensional structure revealed an unusual asymmetric packing of a dimer of two antiparallel right-handed intertwined coiled alpha-helices. The A and B forms exchange on a timescale of seconds by a mechanism that probably involves a relative sliding of the two coiled coils. The amide proton solvent exchange rates demonstrate a stable tetrameric structure. The essential role of Tyr 78 in oligomerization of Mnt, found by previous mutagenesis studies, can be explained by the many hydrophobic and hydrogen bonding interactions that this residue participates in with adjacent monomers.