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R W Hartley

Publications and source records attributed to R W Hartley.

At least 37 records · Page 2Linked to original sources
PubMed1994

Mutational analysis of the active site of RNase of Bacillus intermedius (BINASE).

To elucidate the functional role of some residues in the active site of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant His101 Glu is 2.0-2.7% of that for the native enzyme. The decrease in activity is determined mainly by the decrease in molecular rate constant kcat, with almost unchanged affinity of the enzyme for the substrate, characterized by KM. This is the expected result if His101 acts as an general acid, donating a proton to the leaving group on cleavage of a phosphodiester bond. The replacement of Lys26 by Ala causes a reduction in the enzyme activity to 13-33%, depending on the substrate. The activity decreases are due to changes in both kcat and KM for poly(A) and poly(A) but in kcat alone for GpA. In the latter case the effect is far less than that seen in the homologous mutation in the closely related enzyme, barnase.

Bacillus↗
PubMed1994

Thermodynamics of barnase unfolding.

The thermodynamics of barnase denaturation has been studied calorimetrically over a broad range of temperature and pH. It is shown that in acidic solutions the heat denaturation of barnase is well approximated by a 2-state transition. The heat denaturation of barnase proceeds with a significant increase of heat capacity, which determines the temperature dependencies of the enthalpy and entropy of its denaturation. The partial specific heat capacity of denatured barnase is very close to that expected for the completely unfolded protein. The specific denaturation enthalpy value extrapolated to 130 degrees C is also close to the value expected for the full unfolding. Therefore, the calorimetrically determined thermodynamic characteristics of barnase denaturation can be considered as characteristics of its complete unfolding and can be correlated with structural features--the number of hydrogen bonds, extent of van der Waals contacts, and the surface areas of polar and nonpolar groups. Using this information and thermodynamic information on transfer of protein groups into water, the contribution of various factors to the stabilization of the native structure of barnase has been estimated. The main contributors to the stabilization of the native state of barnase appear to be intramolecular hydrogen bonds. The contributions of van der Waals interactions between nonpolar groups and those of hydration effects of these groups are not as large if considered separately, but the combination of these 2 factors, known as hydrophobic interactions, is of the same order of magnitude as the contribution of hydrogen bonding.

Bacterial Proteins↗
PubMed1994

An improved system for ribonuclease Ba expression.

The extracellular ribonuclease from Bacillus amyloliquefaciens (barnase, RNase Ba) is a well-characterized enzyme extensively used in structure-function studies. A new system for efficient expression and purification of barnase has been developed. The strong regulated expression cassette with the Pr promoter of lambda phage and the cooperative expression of barnase and barstar under its control have been applied to expression of these proteins in Escherichia coli. The expression cassette containing the Pr promoter of E. coli lambda phage under cI repressor regulation and the nucleotide sequence coding for barnase and barstar structural genes were merged into the plasmid pTN441, which was used for large-scale barnase production. The phoA signal peptide was used to express the target protein into cell periplasm. The purification of RNase Ba was carried out in two steps: the initial sample was concentrated followed by RP-HPLC. The system provides a stable yield of homogeneous protein of about 100-150 mg per liter of culture medium.

Amino Acid Sequence↗
PubMed1993

Recognition between a bacterial ribonuclease, barnase, and its natural inhibitor, barstar.

BACKGROUND: Protein-protein recognition is fundamental to most biological processes. The information we have so far on the interfaces between proteins comes largely from several protease-inhibitor and antigen-antibody complexes. Barnase, a bacterial ribonuclease, and barstar, its natural inhibitor, form a tight complex which provides a good model for the study and design of protein-protein non-covalent interactions. RESULTS: Here we report the structure of a complex between barnase and a fully functional mutant of barstar determined by X-ray analysis. Barstar is composed of three parallel alpha-helices stacked against a three-stranded parallel, beta-sheet, and sterically blocks the active site of the enzyme with an alpha-helix and adjacent loop. The buried surface in the interface between the two molecules totals 1630 A2. The barnase-barstar complex is predominantly stabilized by charge interactions involving positive charges in the active site of the enzyme. Asp39 of barstar binds to the phosphate-binding site of barnase, mimicking enzyme-substrate interactions. CONCLUSION: The phosphate-binding site of the enzyme is the anchor point for inhibitor binding. We propose that this is also likely to be the case for other ribonuclease inhibitors.

Bacterial Proteins↗
PubMed1993

Polyethenoadenosine phosphate as a fluorogenic substrate for barnase.

Polyethenoadenosine phosphate is rapidly hydrolyzed by barnase with an accompanying large increase in fluorescence. At pH 8, 0.2 M ammonium acetate, the initial rate of fluorescence increase is proportional to barnase concentration and thus provides an excellent quantitative assay for its activity. The preparation and use of this substrate in the barnase assay is presented in detail.

Bacterial Proteins↗
PubMed1993

Increase of specificity of RNase from Bacillus amyloliquefaciens (barnase) by substitution of Glu for Ser57 using site-directed mutagenesis.

Bacterial ribonucleases from Bacillus amyloliquefaciens and Bacillus intermedius show the specificity towards the nature of a nucleoside at the O3' end of the phosphodiester bond to be split in the preference order G > A >> U > C in the cleavage reactions of polynucleotides. It follows from the X-ray data that the substrate guanosine base is bound at the active site of these RNases in the same manner as for high-specificity guanylic RNases. We supposed that the difference in specificity for the two types of RNases is due to the additional hydrogen bond between the protein and a purine base in the case of bacterial guanyl-preferring RNases in contrast to the high-specificity guanylic RNases. To examine this hypothesis we prepared the Ser57 --> Glu mutant of B. amyloliquefaciens, in which this hydrogen bond is eliminated. Kinetic studies demonstrate that the specificity of this mutant towards guanylic substrates is 35-times greater than that of the wild-type RNases from B. amyloliquefaciens and close to that of RNases T1.

Bacillus↗
PubMed1993

Directed mutagenesis and barnase-barstar recognition.

Directed mutagenesis has been applied to the cloned genes of barnase and barstar, the extracellular ribonuclease of Bacillus amyloliquefaciens and its intracellular inhibitor, to locate residues involved in the mutual recognition of these two proteins. Arg59 and His102 of barnase and Asp35 and Asp39 of barstar have been so identified. With both Cys40 and Cys82 mutated to alanines, barstar is still produced in high yield and is functional both in vitro and in vivo. Methods devised for determining relative and absolute dissociation coefficients for various combinations of mutant and wild-type proteins have allowed us to determine a dissociation coefficient for the complex of wild-type barnase and barstar of about 10(-13) M, with off and on rate constants of 10(-5) s-1 and 10(8) M-1 s-1, respectively.

Bacterial Proteins↗
PubMed1993

Crystallization and preliminary X-ray investigation of barstar, the intracellular inhibitor of barnase.

Crystals of barstar, the intracellular inhibitor of the extracellular ribonuclease produced by Bacillus amyloliquefaciens (barnase), were obtained through vapor phase equilibration using the hanging drop technique. Three crystal forms have been characterized. Forms I and II, crystallized either in potassium phosphate or sodium citrate, are tetragonal; they exhibit a superstructure along the c-axis. Form III crystals, suitable for a high resolution structure determination, were grown from 55-65% ammonium sulfate. This crystal form is hexagonal and diffracts to at least 2 A resolution at a synchrotron radiation source. It belongs to the hexagonal space group P6, with unit cell dimensions a = b = 143.6 A, c = 35.6 A. There are four molecules of barstar in the asymmetric unit. X-ray data have been collected to 2.2 A Bragg spacing. The structure determination is underway in order to analyze conformational changes of barstar upon complexation with barnase.

Bacillus↗
PubMed1993

CT appearances of haematomas in the corpus callosum in patients with subarachnoid haemorrhage.

Corpus callosum haematoma is a rare feature in subarachnoid haemorrhage (SAH), which may result from aneurysms of the anterior communicating artery (ACoA) or pericallosal artery (PCA). In 348 patients with aneurysmal SAH, bleeding from ACoA aneurysms in 88 cases produced no abnormality on CT in 7. Blood in the cistern of the lamina terminalis was the most frequent abnormality (76/88); haematomas of the septum pellucidum, confined to patients with ACoA aneurysms, were seen in 26 (30%). Rupture of PCA aneurysms in 12 patients gave rise to blood in the pericallosal cistern, anterior interhemispheric fissure and cistern of the lamina terminalis in 11. There was no blood in the septum pellucidum or the ventricular system in any case, but haematomas in the corpus callosum occurred in 8 (67%). In all of these, blood extended into the anterodorsal aspect of the callosum and spread posteriorly along its dorsal border. An identical, supracallosal pattern was seen in 2 patients (2.5%) with ACoA aneurysms, in whom haemorrhage was more extensive, with a large frontal lobe haematoma extending up from the cistern of the lamina terminalis in 1 and a haematoma of the septum pellucidum, with intraventricular extension in the other. In 8 patients (9%) with ACoA aneurysms a corpus callosum haematoma appeared to result from passage of blood up through the cistern of the lamina terminalis into the septum pellucidum and thence into the ventral aspect of the anterior corpus callosum; blood was present within the cistern, the septum and the ventricles.

Aneurysm, Ruptured↗
PubMed1993

Comparative study of thermostability and structure of close homologues--barnase and binase.

Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase in the pH region 2-6 have been determined. The barnase heat denaturation (pH 2.8-5.5) proceeds according to the "all-or-none" principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 degrees C at pH 5 to 7 degrees C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5-5.5, but as far as pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp94 by the His 18 residue, absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moments distribution, have been found.

Bacterial Proteins↗
PubMed1992

Towards a new method of assessing the antithrombotic properties of pharmaceutical compounds.

This paper describes the initial development of a novel technique which, following further work, may prove suitable for the in vivo screening of pharmaceutical compounds for evidence of antithrombotic activity. Its inception is shown to be a consequence of an amalgamation of those ultrasonic methods associated with the clinical detection of occlusions and laser Doppler velocimetry. Both topics are discussed and then brought together through a concept whereby the efficacy of compounds might be evaluated in animal models by measuring the velocity of blood in the fluid jet formed distal to an induced thrombus. In accord with the evolved measurement rationale there then follows a description of a laser Doppler microscope, an analyser for extracting the required velocity information from the Doppler shift spectrum and a series of experiments using a thrombus analogue in a narrow-bore glass flow tube. Finally, the results from one in vivo experiment are presented. Speculation about how the method might be developed further is contained within the discussion.

Animals↗
PubMed1991

A novel method for the evaluation of bronchoactive agents in the conscious guinea pig.

We describe a simple, noninvasive, nontraumatic and reproducible method in which the activities of bronchoactive agents may be recorded in six conscious guinea pigs simultaneously. The method involves the use of "head out" whole body plethysmographs from which respiratory rate can be recorded, by monitoring respiration-related changes in pressure within the body chamber. Exposure of a guinea pig to an aerosolised bronchoconstrictor agent causes an increase in respiratory rate, which is quantified by measuring the area under the respiratory rate curve using a purpose-built respiratory computer. This can be carried out for six animals simultaneously and independently. When exposed to a standard bronchoconstrictor aerosol challenge at intervals over a 6 hr period, the areas under the respiratory rate curves for each animal are highly reproducible. Inhalation of nebulized solutions of acetylcholine (ACh), histamine (Hist), 5-hydroxytryptamine, bradykinin, leukotriene D4 and the thromboxane A2-mimetic, U-46619, but not prostaglandin F2 alpha (PGF2 alpha) caused dose-related bronchoconstriction observed as increases in respiratory rate. In addition, salbutamol, clenbuterol, N-ethylcarboxamide adenosine (NECA) and PGE2 all inhibited ACh (1 mg mL-1) and Hist (1 mg mL-1)-induced increases in respiratory rate in a dose-related fashion. The method described, which is both noninvasive and nontraumatic, may therefore be used to quantify in the conscious guinea pig, both bronchoconstrictor and bronchodilator agents.

Administration, Inhalation↗
PubMed1989

Barnase and barstar: two small proteins to fold and fit together.

Barnase and barstar are the extracellular ribonuclease and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Both are small single-chain proteins and thus are suitable for application to the study of how a protein's sequence directs its fold. Barnase has neither disulfide bonds nor non-peptide components and unfolds reversibly in what closely approximates a two-state reaction. The genes for both these proteins have been cloned in E. coli. Expression of barstar is necessary to counter the lethal effect of expressed active barnase. Site-directed mutagenesis is being used to answer specific and general questions relating to protein folding and protein-protein interaction.

Amino Acid Sequence↗
PubMed1989

Translation and processing of Bacillus amyloliquefaciens extracellular RNase.

Bacillus amyloliquefaciens extracellular RNase has been previously cloned and expressed in Bacillus subtilis. Site-specific mutagenesis experiments have identified codon -39 as the start site of translation. We have determined the primary signal peptide cleavage site of preprobarnase and propose a pathway for the conversion of probarnase to mature barnase.

Amino Acid Sequence↗
PubMed1988

Barnase and barstar. Expression of its cloned inhibitor permits expression of a cloned ribonuclease.

Barnase is the extracellular ribonuclease of Bacillus amyloliquefaciens and barstar its specific intracellular inhibitor. The gene for barstar has now been cloned and sequenced. When the wild-type gene for barnase is reconstructed from its previously cloned parts on the same plasmid as the barstar gene, the lethal effect of its expression is suppressed. A plasmid has been devised which directs the secretion of 100 mg per active barnase liter by Escherichia coli and another which provides large (500 to 1000 mg/l) yields of barstar. The structure of these plasmids and the derived 89 amino acid sequence of barstar are reported.

Amino Acid Sequence↗
PubMed1987

Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation.

An inactivated gene for Bacillus amyloliquefaciens extracellular ribonuclease (barnase) has previously been cloned and sequenced following transposon mutagenesis. The intact gene could not be assembled in Escherichia coli and is presumed to be lethal. Therefore, we introduced specific mutations into the barnase gene to prevent its lethal effect. A Gln-73 mutant gene was stable in E. coli but only produced low amounts of barnase antigen. Mutants containing Asp, Gln or Arg, instead of His-102, at the active site were identified by immunological screening for barnase antigen. None of the mutant proteins with alterations at aa residue 102 possessed RNase activity. The level of barnase (Asp-102) was higher in E. coli than in B. subtilis but the protein was not processed to the correct size in E. coli. To obtain correct processing, the barnase (Asp-102) structural gene was fused to the E. coli alkaline phosphatase promoter and signal sequence (phoA). Cells containing this construct secreted correctly processed barnase (Asp-102) into the periplasmic space and culture supernatant at a level of 20 mg/l. Barnase (Asp-102) was purified and found to have an identical N-terminus and a thermal unfolding curve that was nearly identical to that of active barnase (His-102). The cloning and expression of barnase in E. coli will allow detailed analysis of barnase protein folding by molecular genetic approaches.

Alkaline Phosphatase↗