The Genetic and biochemical evidence suggests that DnaJ functionally and physically interacts with DnaK even though no stable binary complex of these two proteins has been observed. DnaJ and DnaK cooperate in many cellular processes, including DNA replication (1-3), protein export (4), and stress response (5, 6). DnaJ and DnaK promote folding of denatured or partially unfolded proteins (7, 8) as well as newly synthesized polypeptides (9). DnaJ can act as a chaperone on its own (6, 8), or it can work together with DnaK (10). DnaK possesses a weak intrinsic ATPase activity (2), and ATP binding and hydrolysis are associated with conformational changes in DnaK that regulate substrate binding and release (11,12). DnaJ stimulates ATP hydrolysis by DnaK (13), and DnaK (MgADP) has the highest affinity toward polypeptide substrates (14, 15).The interaction between DnaJ and DnaK appears to be bimodal. A truncated DnaJ molecule consisting of the Jdomain and Gly͞Phe region stimulates the ATPase activity of DnaK and supports bacteriophage DNA replication, although a higher concentration of the truncated protein is required (16,17). The J-domain by itself neither stimulates the ATPase activity of DnaK nor supports replication of bacteriophage DNA. However, the J-domain plus a peptide added in trans act synergistically to stimulate DnaK's ATPase activity 200-fold (17).Point mutations in the J-domains of DnaJ and DnaJ homologs abolish the ability of these proteins to function with Hsp70 proteins. These mutations include H33Q in DnaJ (4, 16) and two mutations (18) of sec63p, P156N and D157A, which substitute residues in the conserved tripeptide HPD of the J-domain. An insertion of one amino acid between P34 and D35 in DnaJ also abolishes activity in a bacteriophage DNA replication assay (19). Because the tripeptide has no obvious structural role (20) and because there is a high degree of conservation in the HPD segment, the HPD tripeptide could mediate specific interactions between Hsp40 and Hsp70 proteins.The J-domain-Hsp70 interaction can be subverted by viruses. The J-domain of polyomavirus T antigens is required for viral DNA replication and tissue transformation, possibly by mediating interactions with Hsp70. Simian virus 40 (SV40) large T antigen associates with the constitutively expressed Hsc70 (21), and certain J-domain mutants defective in tissue transformation fail to bind Hsc70 (22). Chimeras constructed by substituting the J-domain of DnaJ with the J-domain from any of several polyomavirus T antigens can replace wild-type DnaJ in vivo (23). Likewise, chimeras of T antigen containing the J-domain from DnaJ or a eukaryotic Hsp40 support SV40 DNA replication (24). Nevertheless, the role of the T antigen J-domain in viral DNA replication has not been elucidated.C-terminal domains of DnaJ-family proteins may confer specificity for substrates and͞or function. The C-terminal domains are less well-conserved than the J-domain (25). Most, but not all, DnaJ homologs contain a domain that binds two Zn 2ϩ atoms, a featur...

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirusinfected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and proteinproduction pipelines are reported. Strategies for coexpression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

One- and two-dimensional NMR, UV absorption experiments, and molecular mechanics calculations were conducted on an oligonucleotide duplex (dGCGAATAAGCG)2 which will be referred to as the T-11-mer. This oligonucleotide forms a duplex that is primarily B-form and contains two adjacent G.A and A.A base pairs and two 3' unpaired guanosines. The adjacent mismatch base pairs have an unusual structure which includes overwinding the helix and stacking with the base from the complementary strand (A4 with A8 and G3 with A7) instead of stacking with the base which is sequential on the strand. The exchangeable and nonexchangeable proton NMR spectra of the duplex have been characterized in H2O and D2O solution at neutral and acidic pH. The duplex is stabilized upon protonation; however, no additional hydrogen bonds are formed. We have observed the amino protons of adenosines A4 and A8 and guanosine G3 as a function of temperature and pH. These amino protons are involved in hydrogen bonds with the purine N3 or N7 acting as acceptors. Through the observation of a variety of NOE signals, the structure of the G.A and A.A mismatch base pairs has been defined.

One- and two-dimensional NMR experiments were carried out on a decamer, d-(CGCTTTTCGC).d(GCGAAAAGCG), and on the same sequence with the addition of an unpaired thymidine, d(CGCTTTTCGC).d(GCGAATAAGCG), which will be referred to as the T-bulge decamer. Evidence from one-dimensional NOE experiments on the exchangeable protons indicates that the unpaired thymidine is extrahelical. This conclusion is also supported by numerous cross-peaks in the two-dimensional NOESY spectrum of the nonexchangeable protons. Assignments for all of the resonances, with the exception of the H5' and H5" resonances, have been made for both oligonucleotide duplexes through the use of 2D NOESY, COSY, and relayed COSY experiments. Temperature dependence of the methyl resonance chemical shifts indicates that the unpaired thymidine shows unusual behavior compared to other thymidines in the duplex. Two-dimensional NOESY experiments carried out from 5 to 35 degrees C indicate the unpaired thymidine remains extrahelical throughout this temperature range. A similar temperature dependence for the methyl chemical shift is found in the corresponding single-strand d(GCGAATAAGCG). The oligo-(dA).oligo(dT) tracts in both the decamer and the T-bulge decamer have structures different from B-form DNA and exhibit NOEs similar to those observed in other oligonucleotides containing A.T tracts. The formation of this unusual A.T tract structure may induce the extrahelical conformation of the unpaired thymidine.

We have isolated an endo-␤-galactosidase designated E-ABase from Clostridium perfringens ATCC 10543 capable of liberating both the A trisaccharide (A-Tri; GalNAc␣133(Fuc␣132)Gal) and B trisaccharide (B-Tri; Gal␣133(Fuc␣132)Gal) from glycoconjugates containing blood group A and B glycotopes, respectively. We have subsequently cloned the gene (eabC) that encodes E-ABase from this organism. This gene was found to be identical to the CPE0329 gene of C. perfringens strain 13, whose product was labeled as a hypothetical protein (Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S., and Hayashi, H. human ovarian cyst glycoprotein were established by NMR spectroscopy. The unique specificity of E-ABase should make it useful for studying the structure and function of blood group A-and B-containing glycoconjugates as well as for identifying other glycosidases belonging to the new GH98 family.