Contribution of hydrogen bonds to protein stability.

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

The history of the tetracyclines.

The history of the tetracyclines involves the collective contributions of thousands of dedicated researchers, scientists, clinicians, and business executives over the course of more than 60 years. Discovered as natural products from actinomycetes soil bacteria, the tetracyclines were first reported in the scientific literature in 1948. They were noted for their broad spectrum antibacterial activity and were commercialized with clinical success beginning in the late 1940s to the early 1950s. The second-generation semisynthetic analogs and more recent third-generation compounds show the continued evolution of the tetracycline scaffold toward derivatives with increased potency as well as efficacy against tetracycline-resistant bacteria, with improved pharmacokinetic and chemical properties. Their biologic activity against a wide spectrum of microbial pathogens and their uses in mammalian models of inflammation, neurodegeneration, and other biological systems indicate that the tetracyclines will continue to be successful therapeutics in infectious diseases and as potential therapeutics against inflammation-based mammalian cell diseases.

The imidato-alkenyllithium route for the synthesis of the isoquinocycline-pyrrolopyrrole substructure.

A convergent and effective synthesis of the pyrrolopyrrole substructure (CDEFG) of the isoquinocyclines is reported. A key step is a tin-lithium exchange of an imidato-alkenyltin compound (a ring G equivalent) and the subsequent acylation with a lactone. The resulting acetal is used successfully for the ring F closure to the pyrrolopyrrole. The sole formation of the isoquinocycline N,O-acetal epimer is in accordance with the proposed mechanism for the isomerization of quinocyclines to isoquinocyclines.

Stereocontrolled synthesis of C1-C17 fragment of narasin via a free radical-based approach.

An efficient synthesis of the C1-C17 western unit of narasin was achieved from (S)-Roche ester. Highlights in our synthesis include the successful exploitation of three stereoselective sequences of Lewis acid mediated reaction followed by free-radical-based hydrogen transfer.

Characterization and disruption of exonuclease genes from Streptomyces aureofaciens B96 and S. coelicolor A3(2).

Streptomyces aureofaciens B96 produces several intra- and extracellular enzymes with deoxyribonuclease activity. According to the sequence of the previously published gene exoSc from S. coelicolor A3(2), the DNA sequence from S. aureofaciens B96 was amplified, cloned and expressed in E. coli. The protein product of exoSa gene, recExoSa, was also an exonuclease with DNAase and 5'-phosphomonoesterase activities at optimum temperature 37 degrees C and pH 8.0. It degraded only linear DNA (chromosomal, double-stranded and single-stranded) and linear plasmid DNA from both ends, with a preference to blunt ends in comparison with overhang ends. The purified enzyme exhibited no RNAase activity. Both exoSc and exoSa genes were interrupted by the apramycin resistance gene; constructed fragments were transformed into particular streptomyces protoplasts. Mutation caused by exoSa disruption in S. aureofaciens chromosome and mutation by interrupted exoSc in S. coelicolor were lethal.

Antitumor activity of 4-arylcoumarins from endophytic Streptomyces aureofaciens CMUAc130.

In a search for antitumor agents, we carried out a screening of 4-arylcoumarins isolated from endophytic Streptomyces aureofaciens CMUAc130, by examining their possible inhibitory effect on the growth of s.c. transplanted Lewis lung carcinoma (LLC) in BDF-1 mice by intraperitoneal (i.p.) administration. The 4-arylcoumarins showed antitumor activity with T/C values of 80.8 and 50.0% at doses of 1 and 10 mg/kg of 5,7-dimethoxy-4-p-methoxylphenylcoumarin treatment, respectively and 81.5 and 44.9% at doses of 1 and 10 mg/kg of 5,7-dimethoxy-4-phenylcoumarin treatment, respectively, compared to adriamycin, which was used a positive control, with T/C value of 55.9% at 2 mg/kg. Furthermore, we investigated the possible effects of these compounds on expression of the bcl-2 and Bax oncoproteins in A427, a human lung cancer cell lines. The cells were cultured in vitro for 24 h in RPMI 1640 with 1.5% (v/v) ethanol, 100 microg/ml 5,7-dimethoxy-4-p-methoxylphenylcoumarin or 5,7-dimethoxy-4-phenylcoumarin. Viability was determined by an MTT assay. Total protein was extracted from cell lysates and the bcl-2 and Bax oncoproteins were identified. Western blotting showed a decrease in bcl-2 and an increase in Bax in A427 cell cultured with 5,7-dimethoxy-4-p-methoxylphenylcoumarin or 5,7-dimethoxy-4-phenylcoumarin. We conclude that 5,7-dimethoxy-4-phenylcoumarin is a more potent inhibitor of cell proliferation than 5,7-dimethoxy-4-p-methoxylphenylcoumarin and has more marked effects on oncoprotein expression.

In vitro anti-inflammatory activity of 4-arylcoumarins from endophytic Streptomyces aureofaciens CMUAc130 in murine macrophage RAW 264.7 cells.

This research was undertaken to find the in vitro inflammatory action of 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin produced by Streptomyces aureofaciens CMUAc130. We investigated the effects of 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin not only on the formation of nitric oxide (NO), PGE(2), TNF-alpha, IL-6 and IL-1beta, but also on inducible NO synthase and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells. The data obtained were consistent with the modulation of iNOS enzyme expression. A similar fashion was also observed when LPS-induced PGE(2) release and COX-2 expression were tested. The significant inhibitory effects were shown in concentration-dependent manners. In addition, 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin also mildly but significantly reduced the formation of TNF-alpha. These findings support the application of 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin as anti-inflammatory agents.

Anti-inflammatory activity of 4-arylcoumarins from endophytic Streptomyces aureofaciens CMUAc130 in murine macrophage RAW 264.7 cells.

This research was undertaken to test the in vitro anti-inflammatory action of 5,7,4'-trimethoxy-4-phenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin produced by Streptomyces aureofaciens CMUAc130. The effects of the two coumarins were investigated on the formation of NO, PGE2, and TNF-alpha and also on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells. The data obtained were consistent with the modulation of iNOS enzyme expression. A similar effect was also observed when LPS-induced PGE2 release and COX-2 expression were tested. The inhibitory effects were shown in concentration-dependent manners. The 5,7,4'-Trimethoxy-4-phenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin also mildly but significantly reduced the formation of TNF-alpha.

Biosynthetic gene cluster for the polyenoyltetramic acid alpha-lipomycin.

The gram-positive bacterium Streptomyces aureofaciens Tü117 produces the acyclic polyene antibiotic alpha-lipomycin. The entire biosynthetic gene cluster (lip gene cluster) was cloned and characterized. DNA sequence analysis of a 74-kb region revealed the presence of 28 complete open reading frames (ORFs), 22 of them belonging to the biosynthetic gene cluster. Central to the cluster is a polyketide synthase locus that encodes an eight-module system comprised of four multifunctional proteins. In addition, one ORF shows homology to those for nonribosomal peptide synthetases, indicating that alpha-lipomycin belongs to the classification of hybrid peptide-polyketide natural products. Furthermore, the lip cluster includes genes responsible for the formation and attachment of d-digitoxose as well as ORFs that resemble those for putative regulatory and export functions. We generated biosynthetic mutants by insertional gene inactivation. By analysis of culture extracts of these mutants, we could prove that, indeed, the genes involved in the biosynthesis of lipomycin had been cloned, and additionally we gained insight into an unusual biosynthesis pathway.

Total synthesis of rutamycin B, a macrolide antibiotic from Streptomyces aureofaciens.

Rutamycin B (2) was synthesized from three principal subunits, spiroketal 75, keto aldehyde 83, and aldehyde 108. First, triol 62 was assembled by Julia coupling of sulfone 56 with aldehyde 58 followed by an acid-catalyzed spiroketalization. The three hydroxyl functions of 62 were successfully differentiated, leading to phosphonate 75. The latter was condensed in a Wadsworth-Emmons reaction with 83, prepared in six steps from (R)-aldehyde 76, to give 92. Coupling of the titanium enolate of 92 with 108 afforded Felkin product 109 with high stereoselectivity in a process that is critically dependent on the presence of the p-methoxybenzyl ether in the aldehyde. Transformation of 109 via aldehyde 116 to vinylboronate 122 was followed by macrocyclization under Suzuki conditions to yield 123. Exhaustive desilylation of the latter yielded rutamycin B.

Conformational stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3.

Ribonucleases Sa, Sa2, and Sa3 are three small, extracellular enzymes produced by different strains of Streptomyces aureofaciens with amino acid sequences that are 50% identical. We have studied the unfolding of these enzymes by heat and urea to determine the conformational stability and its dependence on temperature, pH, NaCl, and the disulfide bond. All three of the Sa ribonucleases unfold reversibly by a two-state mechanism with melting temperatures, Tm, at pH 7 of 48.4 degrees C (Sa), 41.1 degrees C (Sa2), and 47.2 degrees C (Sa3). The Tm values are increased in the presence of 0.5 M NaCl by 4.0 deg. C (Sa), 0.1 deg. C (Sa2), and 7.2 deg. C (Sa3). The Tm values are decreased by 20.0 deg. C (Sa), 31.5 deg. C (Sa2), and 27.0 deg. C (Sa3) when the single disulfide bond in the molecules is reduced. We compare these results with similar studies on two other members of the microbial ribonuclease family, RNase T1 and RNase Ba (barnase), and with a member of the mammalian ribonuclease family, RNase A. At pH 7 and 25 degrees C, the conformational stabilities of the ribonucleases are (kcal/mol): 2.9 (Sa2), 5.6 (Sa3), 6.1 (Sa), 6.6 (T1), 8.7 (Ba), and 9.2 (A). Our analysis of the stabilizing forces suggests that the hydrophobic effect contributes from 90 to 110 kcal/mol and that hydrogen bonding contributes from 70 to 105 kcal/mol to the stability of these ribonucleases. Thus, we think that the hydrophobic effect and hydrogen bonding make large but comparable contributions to the conformational stability of these proteins.

Purification and partial characterization of two thioredoxins from Streptomyces aureofaciens.

Thioredoxins are low molecular weight proteins, which participate in a wide spectrum of biochemical reactions. Two thioredoxins from Streptomyces aureofaciens 3239 have been purified to homogeneity by a sequence of chromatography steps including chromatography on Sephacryl S-300, Phenyl Sepharose CL 4B and MonoQ HR 5/5. Thioredoxin activity clearly separates into two protein fractions on MonoQ HR 5/5 chromatography. Molecular weights determined by chromatography on Superose 12 HR 10/30 and sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed M(r) approximately 10,500 for thioredoxin 1 (TR1) and M(r) approximately 11,000 for thioredoxin 2 (TR2). The isoelectric points of the two thioredoxins are different pI = 4.7 for TR1 and 5.6 for TR2, respectively. Both were effectively reduced with NADPH in reaction catalyzed by Streptomyces aureofaciens thioredoxin reductase. The specific activity of viewly for discovered TR2 is about 1/4 of the specific activity of TR1. Both thioredoxins activate spinach NADPH-malate dehydrogenase. Activation of this enzyme by TR2 is only half effective than by TR1. The stability of TR1 is high and similar to thioredoxins from other organisms unlike the activity of TR2 which is decreased during purification. The proteins diversed in their contents in exponentially growing mycelium.

Data handling in quantitative microanalysis in biology.

In cell biology, electron probe X-ray microanalysis can reveal the distribution of chemical elements inside a single cell. The full description of a biological system (cell population, tissue) requires a great number of spot measurements. In quantitative analysis, the measurements are subject to experimental errors of several types; moreover, the relations between the resulting values are usually more interesting than the absolute concentrations. Nevertheless, the proper evaluation of quantitative values can discover information more on the object of study. A system of simple statistical tests is suggested here which can solve several problems. Some concentration values can be far from the statistical average due to errors in measurement; therefore, a statistical test of plausibility of the measured values is carried out. In the compartments (e.g., nucleus, cytoplasm or other selected areas), the distribution of an element can be nonhomogeneous, and hence a statistical test of homogeneity of the element distribution in specified areas is provided. The tests continue with a test for correlation, in which the concentrations of a given element in a pair of specified areas are compared. These test proceed step-by-step for all elements of interest. Subsequently, the relations of concentrations in all possible pairs of elements in the area in question are calculated. Moreover, cells within a population can be different from the point of view of elemental concentration; a statistical test of homogeneity of the cell population is provided. In the case of nonhomogeneity, the concentration values and/or cells within a population are clustered into homogeneous groups. The evaluation is carried out automatically, with a simple program. The system of programs, in which the program for evaluation is incorporated, is included semi-on-line in the EDAX9900 system, where the measurement and evaluation are carried out in sequence. The results for a population of Streptomyces aureofaciens are shown as an example.

Quantitative analysis of gel electrophoretograms by image analysis and least squares modeling.

A computer-aided quantitative method for a complex analysis of gel electrophoretograms is presented. The analysis consists of several steps: (i) determination of the background image by methods of mathematical morphology and its subtraction from the gel image, (ii) selection of an appropriate part of the gel lane including curved lanes and lanes with a nonuniform width, (iii) computation of the lane densitogram by averaging several lane-parallel scans, (iv) decomposition of the lane densitogram into component bands using a data selecting algorithm and Marquardt's minimizer. Several different functions for component bands are utilized. It is shown that the densitogram can be decomposed into component bands with reasonable accuracy only if an appropriate model function is chosen. The algorithms are tested on several different gel electrophoretograms which show typical features as a nonuniform background, curved lanes, an asymmetrical band shape and a superposition of small bands on the shoulders of big ones. It is shown that overlapped bands are best approximated by an asymmetrical Gausian curve and an asymmetrical Gauss-Cauchy function. Linear response to the serial dilution of the protein sample is tested.

Isolation and characterization of glycocalyx in Streptomyces aureofaciens.

The surface layer, considered to be glycocalyx according to electron-microscopic observations, was separated from a low-production strain of Streptomyces aureofaciens by solubilization with urea and subsequent sonication. The isolation procedure was developed using various agents; neutral phosphatase served as a marker indicating the amount of the material released. The peripheral structure consisted predominantly of glycoprotein and differed from S-layers.