Screening for novel antimicrobials from encoded combinatorial libraries by using a two-dimensional agar format.

A sensitive lawn-based format has been developed to screen bead-tethered combinatorial chemical libraries for antimicrobial activity. This method has been validated with beads linked to penicillin V via a photocleavable chemical linker in several analyses including a spike-and-recover experiment. The lawn-based screen sensitivity was modified to detect antibacterial compounds of modest potency, and a demonstration experiment with a naive combinatorial library of over 46,000 individual triazines was evaluated for antibacterial activity. Numerous hits were identified, and both active and inactive compounds were resynthesized and confirmed in traditional broth assays. This demonstration experiment suggests that novel antimicrobial compounds can be easily identified from very large combinatorial libraries of small, nonpeptidic compounds.

Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound to wild type and mutant ATPase fragment.

The ATPase fragment of the bovine 70-kDa heat shock cognate protein is an attractive construct in which to study its mechanism of ATP hydrolysis. The three-dimensional structure suggests several residues that might participate in the ATPase reaction. Four acidic residues (Asp-10, Glu-175, Asp-199, and Asp-206) have been individually mutated to both the cognate amine (asparagine/glutamine) and to serine, and the effects of the mutations on the kinetics of the ATPase activity (Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12893-12898) and the structure of the mutant ATPase fragments have been determined, typically to approximately 2.4 A resolution. Additionally, the structures of the wild type protein complexed with MgADP and Pi, MgAMPPNP (5'-adenylyl-beta, gamma-imidodiphosphate) and CaAMPPNP have been refined to 2.1, 2.4, and 2.4 A, respectively. Combined, these structures provide models for the prehydrolysis, MgATP-bound state and the post-hydrolysis, MgADP-bound state of the ATPase fragment. These models suggest a pathway for the hydrolytic reaction in which 1) the gamma phosphate of bound ATP reorients to form a beta, gamma-bidentate phosphate complex with the Mg2+ ion, allowing 2) in-line nucleophilic attack on the gamma phosphate by a H2O molecule or OH- ion, with 3) subsequent release of inorganic phosphate.

Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. I. Kinetic analyses of active site mutants.

The 70-kDa heat shock cognate protein is a member of a highly conserved family of molecular chaperones in which the binding and release of target polypeptides are coupled to the chaperones' ATPase activity. The ATPase activity resides in the amino-terminal 44-kDa fragment of the protein. Four acidic residues of the ATPase fragment which might participate in catalysis (Asp-10 and Asp-199, which are Mg2+ ion ligands; Glu-175 and Asp-206, which are candidates for a role as catalytic base) have been individually mutated to both the cognate amide residue (aspartate to asparagine, glutamate to glutamine) and to serine, and the effects of the mutations on the kinetics (this manuscript) and structure (Flaherty, K.M., Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12899-12907) have been determined. Changes at Asp-10 and Asp-199 reduced kcat to approximately 1% of wild type; changes at Glu-175 and Asp-206 reduced kcat to approximately 10% of wild type. Changes to Asp-199 and Asp-206 had little effect on Km; changes to Asp-10 and Glu-175 increased Km 10-100-fold. These data suggest that the bound magnesium ion and its local environment are crucial to catalysis; they argue against a single residue acting as the sole essential general base catalyst in the hydrolytic reaction.

Transgenic corn plants expressing MDMV strain B coat protein are resistant to mixed infections of maize dwarf mosaic virus and maize chlorotic mottle virus.

The maize dwarf mosaic virus strain B (MDMV-B) coat protein (cp) gene was cloned into a monocot expression cassette and introduced into sweet corn cell suspension cultures via particle bombardment or electroporation. Transformed cells were selected on culture media containing 300 mg/l kanamycin, and plants were regenerated. Cells from all transformed lines expressed the cp gene; and one transgenic line synthesized approximately 100-200 micrograms MDMV-cp per gram fresh weight. Plants regenerated from this line were challenged with a virus inoculum concentration adjusted to produce symptoms in nontransgenic controls at six days post inoculation. In growth chamber studies, the presence of the MDMV-cp provided resistance to inoculations with MDMV-A or MDMV-B and to mixed inoculations of MDMV and maize chlorotic mottle virus.

Crystals of a hammerhead ribozyme.

Hammerhead ribozyme-inhibitor complexes consisting of an RNA "enzyme" strand and a DNA "inhibitor" strand have been crystallized in four different crystal forms. The crystal form that is most attractive for structure determination diffracts to approximately 3.2 A resolution; it is trigonal, space group P3(1)21 or enantiomorph, a = 92.5 A, c = 185.0 A.

Uncoating protein (hsc70) binds a conformationally labile domain of clathrin light chain LCa to stimulate ATP hydrolysis.

Uncoating of clathrin-coated vesicles is mediated by the heat shock cognate protein, hsc70, and requires clathrin light chains (LCa and LCb) and ATP hydrolysis. We demonstrate that purified light chains and synthetic peptides derived from their sequences bind hsc70 to stimulate ATP hydrolysis. LCa is more effective than LCb in stimulating hsc70 ATPase and in inhibiting clathrin uncoating by hsc70. These differences correlate with high sequence divergence in the proline- and glycine-rich region (residues 47-71) that forms the hsc70 binding site. For LCa, but not LCb, this region undergoes reversible conformational changes upon perturbation of the ionic strength or the calcium ion concentration. Our results show that LCa is more important for interactions with hsc70 than is LCb and suggest a model in which the LCa conformation regulates coated vesicle uncoating.

Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein.

The three-dimensional structure of the amino-terminal 44K ATPase fragment of the 70K bovine heat-shock cognate protein has been solved to a resolution of 2.2 A. The ATPase fragment has two structural lobes with a deep cleft between them; ATP binds at the base of the cleft. Surprisingly, the nucleotide-binding 'core' of the ATPase fragment has a tertiary structure similar to that of hexokinase, although the remainder of the structures of the two proteins are completely dissimilar, suggesting that both the phosphotransferase mechanism and the substrate-induced conformational change intrinsic to the hexokinases may be used by the 70K heat shock-related proteins.

Crystals of an ATPase fragment of bovine clathrin uncoating ATPase.

A 44,000 Mr amino-terminal, clathrin-independent ATPase fragment of the bovine clathrin uncoating ATPase has been crystallized in a form suitable for X-ray diffraction studies. The crystals are orthorhombic, space group P2(1)2(1)2(1), a = 145.3 A, b = 65.0 A, c = 46.9 A, with one protein molecule per asymmetric unit (1 A = 0.1 nm).

Hybridization and partial cDNA sequence analyses of bovine lung angiotensin I-converting enzyme.

The mRNA encoding angiotensin I-converting enzyme, a zinc-metallo dipeptidyl carboxyhydrolase, has been identified in extracts prepared from bovine lung tissue. Bovine lung poly(A) + mRNAs were subjected to electrophoresis and northern blot hybridization analysis using a radiolabeled synthetic 24-deoxyoligonucleotide probe complementary to eight codons for amino acids at the active-site of the enzyme (Harris, R.B. & Wilson, I.B., J. Biol. Chem. 260, 2208-2211, 1985). This amino acid sequence contains the catalytic glutamic acid residue. A single RNA species (approximately equal to 4 kb) was detected which is 1 kb larger than predicted from the molecular weight of the enzyme. The excess nucleic acid composition may be due to leader and/or trailer sequences or the RNA may encode a high molecular weight precursor form of the enzyme. We have cloned an EcoR1-HindIII digest fragment (1400 bp) of the duplex cDNA derived from the bovine lung converting enzyme poly(A) + mRNA and also Bal31 deletion fragments generated from the 1400 bp clone. Several of the Bal31 clones contain the active-site sequence codons of the enzyme and the complete cDNA sequence of one of these (72 bp) has been determined. We found the amino acid sequence at the active site to be -Phe-Thr-Glu-Leu-Ala-Asn-Ser-, containing the catalytic Glu residue. This sequence is identical with the sequence that we previously determined by manual Edman degradation analysis of the appropriate active-site peptide except that we now find Asn instead of Asp. We have sequenced 670 bp of the 1400 bp clone but have not yet overlapped the active-site sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for two RNA polymerase activities in Euglena gracilis chloroplasts.

Two types of RNA polymerase activity were isolated from Euglena gracilis chloroplasts and compared. One polymerase is tightly bound to chloroplast DNA; this complex is called the transcriptionally active chromosome (TAC) (Rushlow, K. E., Orozco, E. M., Jr., Lipper, C., and Hallick, R. B. (1980) J. Biol. Chem. 255, 3786-3792). The other activity is found in a soluble extract of Euglena chloroplasts. The soluble extract is dependent upon an exogenous DNA template for activity. The two activities can be isolated in two distinct subchloroplast fractions from a single chloroplast preparation. The soluble extract is selective for transcription of transfer RNA genes, whereas the TAC is selective for ribosomal RNA genes. TAC and the soluble extract respond differently to KCl and Mg2+. The soluble extract is sensitive to heparin, and TAC is resistant. The two activities have different temperature optima. Based on this evidence, we conclude that Euglena chloroplasts have at least two distinct RNA polymerase activities.