Crystals of the NC1 domain of human type IV collagen

Crystals of the non-collagenous C-terminal region (NC1) of type IV collagen have been obtained from human placenta. These crystals diffract to 2.0 A, and belong to space group P22(1)2(1), with cell dimensions a = 81 A, b = 158 A, c = 138 A, alpha = beta = gamma = 90 degrees. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.     

Crystallographic refinement and structure of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum at 1.7 A resolution

The amino acid sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum has been fitted to the electron density maps. The resulting protein model has been refined to a nominal resolution of 1.7 A using the constrained-restrained least-squares refinement program of Sussman and the restrained least-squares refinement program of Hendrickson & Konnert. The crystallographic refinement, based on 76,452 reflections with F greater than sigma (F) in the resolution range 5.5 to 1.7 A resulted in a crystallographic R-factor of 18.0%. The asymmetric unit contains one dimeric ribulose-1,5-biphosphate carboxylase molecule, consisting of 869 amino acid residues and 736 water molecules. The geometry of the refined model is close to ideal, with root-mean-square deviations of 0.018 A in bond lengths and 2.7 degrees in bond angles. Two loop regions, comprising residues 54 to 63 and 324 to 335, and the last ten amino acid residues at the C terminus are disordered in our crystals. The expected trimodal distribution is obtained for the side-chain chi 1-angles with a marked preference for staggered conformation. The hydrogen-bonding pattern in the N-terminal beta-sheet and the parallel sheet in the beta/alpha-barrel is described. A number of hydrogen bonds and salt bridges are involved in domain-domain and subunit-subunit interactions. The subunit-subunit interface in the dimer covers an area of 2800 A2. Considerable deviations from the local 2-fold symmetry are found at both the N terminus (residues 2 to 5) and the C terminus (residues 422 to 457). Furthermore, loop 8 in the beta/alpha-barrel domain has a different conformation in the two subunits. A number of amino acid side-chains have different conformations in the two subunits. Most of these residues are located at the surface of the protein. An analysis of the individual temperature factors indicates a high mobility of the C-terminal region and for some of the loops at the active site. The positions and B-factors for 736 solvent sites have been refined (average B: 45.9 A2). Most of the solvent molecules are bound as clusters to the protein. The active site of the enzyme, especially the environment of the activator Lys191 in the non-activated enzyme is described. Crystallographic refinement at 1.7 A resolution clearly revealed the presence of a cis-proline at the active site. This residue is part of the highly conserved region Lys166-Pro167-Lys168.     

3-Diazirine-derivatives of bile salts for photoaffinity labeling

New carbene-generating photolabile bile salt derivatives, 3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oic acid and (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oyl)-2- aminoethanesulfonic acid were synthesized with high specific radioactivity. These 3-diazirine-derivatives could be activated to the corresponding carbenes by irradiation with ultraviolet light at 350 nm with a half-life time of 2 min. The 3-diazirine derivatives behaved in enterohepatic circulation like the natural bile salts. The uptake of [3H]taurocholate into isolated hepatocytes was competitively inhibited by (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2- aminoethanesulfonic acid indicating that the 3,3-azo-derivative of taurocholate shares the hepatic transport systems for natural bile salts. It was demonstrated that the radioactively labeled 3-diazirine bile salt derivatives are useful probes for photoaffinity labeling of bile salt binding proteins especially in intact cells and tissues.     

The molecular basis for Duchenne versus Becker muscular dystrophy: Correlation of severity with type of deletion

About 60% of both Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) is due to deletions of the dystrophin gene. For cases with a deletion mutation, the "reading frame" hypothesis predicts that BMD patients produce a semifunctional, internally deleted dystrophin protein, whereas DMD patients produce a severely truncated protein that would be unstable. To test the validity of this theory, we analyzed 258 independent deletions at the DMD/BMD locus. The correlation between phenotype and type of deletion mutation is in agreement with the "reading frame" theory in 92% of cases and is of diagnostic and prognostic significance. The distribution and frequency of deletions spanning the entire locus suggests that many "in-frame" deletions of the dystrophin gene are not detected because the individuals bearing them are either asymptomatic or exhibit non-DMD/non-BMD clinical features.     

Excess information at bacteriophage T7 genomic promoters detected by a random cloning technique.

In our previous analysis of the information at binding sites on nucleic acids, we found that most of the sites examined contain the amount of information expected from their frequency in the genome. The sequences at bacteriophage T7 promoters are an exception, because they are far more conserved (35 bits of information content) than should be necessary to distinguish them from the background of the Escherichia coli genome (17 bits). To determine the information actually used by the T7 RNA polymerase, promoters were chemically synthesized with many variations and those that function well in an in vivo assay were sequenced. Our analysis shows that the polymerase uses 18 bits of information, so the sequences at phage genomic promoters have significantly more information than the polymerase needs. The excess may represent the binding site of another protein.     

Molecular deletion patterns in Duchenne and Becker type muscular dystrophy

Summary DNA from 80 Duchenne (DMD) and 15 Becker (BMD) index patients was analyzed with 12 genomic probes and the total cDNA. Deletions were detected in 24 DMD (30%) and 10 BMD patients (67%) by genomic probes alone, mostly p20, pXJ, and/or pERT87. All deletions were confirmed by cDNA probes, and an additional 29 DMD deletions were detected, resulting in a total of 63/95 deletions (66%). The majority of the deletions are localized between kb 6.7 and 9.7 of the cDNA; a smaller group, between kb 0.5 and 3.5. Of the deletions, 90% are detected by the three cDNA probes 1–2a, 7, and 8. This can be applied to strategies for carrier detection and prenatal diagnosis. The order of 13 exon-containing HindIII fragments in the region between probes 7 and 9–10, where most of the deletions are found, could be defined. The deletion patterns in DMD and BMD patients are different and well in accordance with the “reading frame theory” of Monaco and coworkers. Thus our findings indicate that a DMD or BMD phenotype may be predicted according to the breakpoint position and the number of deleted exons.     

Expression of synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) in Escherichia coli

Synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) were assembled and cloned using established Escherichia coli expression plasmids. Transformed E. coli cells were able to synthesize either a fusion protein, comprising the first seven amino acids of beta-galactosidase, a linker fragment and bovine FGF, or genomic human bFGF. The two growth factors were purified from E. coli lysates by cation exchange and heparin-Sepharose affinity chromatography. The purified recombinant proteins were biologically active as monitored by their mitogenic activity for bovine aortic endothelial cells and their angiogenic capacity in the rabbit cornea.     

Preliminary crystallographic data for transketolase from yeast

Crystals of the vitamin B1-dependent enzyme transketolase from baker's yeast have been grown from the apo- and the holoform of the enzyme, using PEG as precipitant. The crystals are orthorhombic, space group P2(1)2(1)2(1) with cell constants a = 76.3 A, b = 114.2 A, and c = 163.5 A. The crystals are stable in the x-ray beam and diffract to at least 2.2 A on a conventional x-ray source. The enzyme is a dimer of identical subunits, and a Vm value of 2.2 A/dalton indicates that the asymmetric unit contains a dimer. Rotation function calculations using native data (10-5 A) revealed a local 2-fold rotation axis with phi = 0 degree and omega = 20 degrees.     

Crystal structure of the complex of ribulose-1,5-bisphosphate carboxylase and a transition state analogue, 2-carboxy-D-arabinitol 1,5-bisphosphate

The crystal structure of the binary complex of nonactivated ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and a transition state analogue, 2-carboxy-D-arabinitol 1,5-bisphosphate has been determined to 2.6 A resolution with x-ray crystallographic methods. The transition state analogue binds in a rather extended conformation at the active site. The orientation of the transition state analogue within the active site could be determined from the electron density maps. The P1 phosphate group of the analogue binds at a site built up of residues from loops 5 and 6 of the alpha/beta-barrel. The phosphate group interacts with the side chains of the conserved residues Arg-288, His-321, and Ser-368 and with main chain nitrogens from residues Thr-322 and Gly-323. The second phosphate group of the transition state analogue binds at the opposite side of the barrel close to loops 1 and 8. Significant differences for the positions and interactions of the P2 phosphate group with the enzyme are found in the two subunits of the dimer. The different mode of binding for this phosphate group in the two subunits is interpreted as a consequence of different conformations of the polypeptide chain observed in loops 6 and 8. The P2 phosphate group interacts with the sidechains of Lys-166 and Lys-329. Loop 6, which is disordered in the nonactivated, nonliganded enzyme is considerably more ordered in one of the subunits, probably due to the interaction of the side chain of Lys-329 with the P2 phosphate group. Almost all oxygen atoms are hydrogen bonded to groups on the enzyme. The carboxyl group forms hydrogen bonds to the side chain of the conserved Asn-111. The binding of the transition state analogue to the nonactivated enzyme is different from the binding of the analogue to activated spinach ribulose-bisphosphate carboxylase.     

Phenolic substances as allelopathic agents arising during the degradation of rye (Secale cereale) tissues

Rye seedlings, tillering plants and crop residues were allowed to decompose in model incubation experiments. Young tissues gave rise to high concentrations of allelochemicals, whereas crop residues did not produce inhibitors. Seven phenolic acids were identified in the investigated materials; p-hydroxybenzoic protocatechuic, gallic, vanillic, syringic, p-coumaric, ferulic as well as benzoic acid. However, neither the level of these acids nor the total content of phenolic compounds corresponded to the level of phytotoxicity determined in bioassays. This demonstrated that, apart from phenolics, other unidentified water-soluble organic compounds were also responsible for the toxicity of rye decomposition products. The study was conducted within program CPBP 04.10.03. The study was conducted within program CPBP 04.10.03.