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C Betzel

Publications and source records attributed to C Betzel.

At least 55 records · Page 3Linked to original sources
PubMed1998

Steady-state and time-resolved fluorescence of Esperase: comparison with the X-ray structure in the region of the two tryptophans.

Fluorescence emission properties of the alkaline protease Esperase have been investigated using steady-state and time-resolved fluorescence spectroscopy. The local polarity and solvent accessibility of the tryptophyl chromophores is characterized. Quenching studies demonstrated that Trp 6 and Trp 113 are 'buried' to acrylamide, iodide ions and caesium ions. An abnormally low tryptophan quantum yield was calculated showing that the emission of the two indole rings is significantly quenched by nearby side chains or peptide bonds. The fluorescence decay of PMS-Esperase was well fitted by two exponentials with lifetimes of 2.7 and 0.35 ns. X-ray data for Esperase (S. Klupsch, Ph.D. Thesis, University of Hamburg, Hamburg, Germany) in the region of the two tryptophans were used to explain the observed emission properties. Gln 182 and Asn 204 as well as Asn 117 and Met 119 are the most likely quenchers, respectively, of the Trp 6 and Trp 113 fluorescence. The two tryptophans in Esperase are 'buried' in hydrophobic regions and are excellent intrinsic probes to study folding-unfolding reactions. Experiments in the presence and absence of added calcium ions demonstrated the stabilizing role of the Ca(2+)-binding sites.

Computer Graphics↗
PubMed1998

Spectroscopic properties and stability of the neurotoxic complex. Vipoxin and its components.

The neurotoxin Vipoxin from the venom of Vipera ammodytes meridionalis is a complex between a toxic basic phospholipase A2 (PLA2) and a non-toxic acidic protein inhibitor (Inh). Tryptophan fluorescence parameters are determined for the complex and for its components. Iodide, caesium and acrylamide are not efficient quenchers of the Vipoxin indole emission. Increased accessibilities of tryptophans to ionic and neutral quenchers are found after the dissociation of the complex. Trp 20 and Trp 31 became more 'exposed' in the separated individuals proteins. The indole rings of the complex are located in a positively charged environment. Inspection of the Vipoxin X-ray model showed that the three tryptophyl side chains are located in the interface region between the enzyme and the inhibitor and are completely 'exposed' in the separated components of the complex. In Vipoxin an efficient 'interchain' energy transfer between tyrosyl and tryptophyl residues from different polypeptide chains occurs. Static quenching with acrylamide is also detected in PLA2 and Inh. The free energy changes deltaG D for the unfolding reactions of Vipoxin, PLA2 and Inh are determined in circular dichroism spectroscopy. The complex formation between the toxic PLA2 and the inhibitor increases deltaG HD2O to 23.5 kJ mol-1.

Acrylamide↗
PubMed1997

Crystal structure of vipoxin at 2.0 A: an example of regulation of a toxic function generated by molecular evolution.

Vipoxin is the main toxic component in the venom of the Bulgarian snake Vipera ammodytes meridionalis, the most toxic snake in Europe. Vipoxin is a complex between a toxic phospholipase A2 (PLA2) and a non-toxic protein inhibitor. The structure is of genetic interest due to the high degree of sequence homology (62%) between the two functionally different components. The structure shows that the formation of the complex in vipoxin is significantly different to that seen in many known structures of phospholipases and contradicts the assumptions made in earlier studies. The modulation of PLA2 activity is of great pharmacological interest, and the present structure will be a model for structure-based drug design.

Amino Acid Sequence↗
PubMed1996

Crystallization and preliminary X-ray analysis of a low density lipoprotein from human plasma.

Single crystals of human plasma low density lipoprotein (LDL), the major transport vehicle for cholesterol in blood, have been produced with a view to analysis of the three-dimensional structure by x-ray crystallography. Crystals with dimensions of approximately 200 x 100 x 50 microm have been reproducibly obtained from highly homogeneous LDL particle subspecies, isolated in the density ranges d = 1.0271-1. 0297 g/ml and d = 1.0297-1.0327 g/ml. Electron microscopic imaging of ultrathin-sectioned preparations of the crystals confirmed the existence of a regular, quasihexagonal arrangement of spherical particles of approximately 18 nm in diameter, thereby resembling the dimensions characteristic of LDL after dehydration and fixation. X-ray diffraction with synchrotron radiation under cryogenic conditions revealed the presence of well resolved diffraction spots, to a resolution of about 29 A. The diffraction patterns are indexed in terms of a triclinic lattice with unit cell dimensions of a = 16. 1 nm, b = 39.0 nm, c = 43.9 nm; alpha = 96.2 degrees, beta = 92.1 degrees, gamma = 102 degrees, and with space group P1.

Crystallography, X-Ray↗
PubMed1996

Crystallization and preliminary diffraction data of a major pollen allergen. Crystal growth separates a low molecular weight form with elevated biological activity.

Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients. In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein. The allergen was expressed in Escherichia coli and subsequently purified. Spontaneous conversion of these preparations to a mixture of various forms with molecular sizes between 10 and 29 kDa was consistently observed. Surprisingly, crystals could be grown from this heterogenous preparation. Single crystals, redissolved and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot, yielded one distinct low molecular weight protein, which was identified by amino acid sequencing as the C-terminal 13-kDa portion of the allergen. Histamine release assays with single crystal solutions using basophils of an allergic patient demonstrated allergenicity comparable with that of the holo-allergen. By contrast, RNase activity of the crystallized C-terminal form was 23 times higher than that of the full-length parent allergen. Crystals were used to collect preliminary diffraction data; the space group was evaluated to I4122 with cell dimensions of a = 87.7 A, b = 87.7 A, and c = 59.6 A. We conclude that preferential crystal growth of the 13-kDa form is indicative of a compact conformation of this particular C-terminal portion of the allergen. Thus, we show here that protein crystallization is not only a prerequisite for structural analyses, but it also can provide a unique separation technique to localize the functional domain of a major allergen.

Allergens↗
PubMed1996

Subtilisin BPN' at 1.6 A resolution: analysis for discrete disorder and comparison of crystal forms.

The three-dimensional structure of the serine protease subtilisin BPN' (SBT) has been refined at 1.6 A resolution in space group C2 to a final R value of 0.17. 17 regions of discrete disorder have been identified and analyzed. Two of these are dual-conformation peptide units; the remainder involve alternate rotamers of side chains either alone or in small clusters. The structure is compared with previously reported high-resolution models of SBT in two other space groups, P2(1)2(1)2(1) and P2(1). Apart from the surface, there are no significant variations in structure among the three crystal forms. Structural variations observed at the protein surface occur predominantly in regions of protein-protein contact. The crystal packing arrangements in the three space groups are compared.

Journal Article↗
PubMed1996

Structure of a ternary complex of proteinase K, mercury, and a substrate-analogue hexa-peptide at 2.2 A resolution.

The crystal structure of a ternary complex of proteinase K, Hg(II) and a hexapeptide N-Ac-Pro-Ala-Pro-Phe-Pro-Ala-NH2 has been determined at 2.2 A resolution and refined to an R factor of 0.172 for 12,910 reflections. The mercury atom occupies two alternate sites, each of which was assigned an occupancy of 0.45. These two sites are bridged by Cys-73 S gamma which forms covalent bonds to both. Both mercury sites form regular polyhedrons involving atoms from residues Asp-39, His-69, Cys-73, His-72, Met-225, and Wat-324. The complex formation with mercury seems to disturb the stereochemistry of the residues of the catalytic triad Asp-39, His-69, and Ser-224 appreciably, thus reducing the enzymatic activity of proteinase K to 15%. The electron density in the difference Fourier map shows that the hexapeptide occupies the S1 subsite predominantly and the standard recognition site constituted by Ser-132 to Gly-136 and Gly-100 to Tyr-104 segments is virtually empty. The hexapeptide is held firmly through a series of hydrogen bonds involving protein atoms and water molecules. As a result of complex formation, Asp-39, His-69, Met-225, Ile-220, Ser-219, Thr-223, and Ser-224 residues move appreciably to accommodate the mercury atoms and the hexapeptide. The largest movement is observed for Met-225 which is involved in multiple interactions with both mercury and the hexapeptide. The activity results indicate an inhibition rate of 95%, as a result of the combined effect of mercury and hexapeptide.

Amides↗
PubMed1996

Strategy to design peptide inhibitors: structure of a complex of proteinase K with a designed octapeptide inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2 at 2.5 A resolution.

The crystal structure of a complex formed by the interaction between proteinase K and a designed octapeptide amide, N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2, has been determined at 2.5 A resolution and refined to an R-factor of 16.7% for 7,430 reflections in the resolution range of 8.0-2.50 A. The inhibitor forms a stable complex through a series of hydrogen bonds and hydrophobic interactions with the protein atoms and water molecules. The inhibitor is hydrolyzed between Phe4I and DAla5I (I indicates the inhibitor). The two fragments are separated by a distance of 3.2 A between the carbonyl carbon of Phe4I and the main-chain nitrogen of DAla5I. The N-terminal tetrapeptide occupies subsites S1-S5 (S5 for acetyl group), whereas the C-terminal part fits into S1'-S5' region (S5' for amide group). It is the first time that such an extended electron density for a designed synthetic peptide inhibitor has been observed in the prime region of an enzyme of the subtilisin family. In fact, the inhibitor fills the recognition site completely. There is only a slight rearrangement of the protein residues to accommodate the inhibitor. Superposition of the present octapeptide inhibitor on the hexapeptide inhibitor studied previously shows an overall homology of the two inhibitors, although the individual atoms are displaced significantly. It suggests the existence of a recognition site with flexible dimensions. Kinetic studies indicate an inhibition rate of 100% by this specifically designed peptide inhibitor.

Amides↗
PubMed1996

Fluorescence decay of tryptophans in serine proteinases from microorganisms: relation to X-ray models.

Fluorescence decay kinetics of indole groups in five proteinases from microorganisms are reported. The data show differences between the excited state lifetimes of the tryptophans located in identical positions in the polypeptide chains of the closely related proteinases mesentericopeptidase and subtilisin Novo. The lifetime of the single Trp 113 in subtilisins DY and Carlsberg are identical. The microenvironments of this residue in the four subtilisins are identical and probably its fluorescence is quenched in these proteins. The crystallographic models of the enzymes investigated were analysed in the region of the tryptophyl residues and provide an explanation for the observed emission properties.

Bacillus↗
PubMed1996

Three dimensional structure of the antibiotic bacitracin A complexed to two different subtilisin proteases: novel mode of enzyme inhibition.

The three dimensional crystal structures of thermitase-bacitracin (TMTBAC), Savinase- bacitracin (SAVBAC) and Savinase-zinc/bacitracin (SAVBAC/ZN) have been determined by X-ray diffraction to 2.2 angstroms, 2.2 angstroms and 1.95 angstroms resolution, respectively. The multifunctional dodecapeptide bacitracin A secreted by Bacillus licheniformis is well known as an antibiotic against gram-positive bacteria but also as an inhibitor for different proteases. The bacteriocidal activity requires the presence of divalent metal cations such as zinc or nickel. It also could be shown that bacitracin A is bound to subtilisin in the Bacillus licheniformis. This complex is stable throughout the purification by chromatography. Therefore the subtilisin proteases thermitase and Savinase were used for cocrystallization with bacitracin A and zinc/bacitracin A. The complexes are formed from two enzyme molecules and two bacitracin A molecules. All three complexes show the same novel mode of enzyme inhibition. Each bacitracin A chain binds non-covalently to two protease molecules: to the catalytic side of one and to the substrate recognition side of the second protease molecule. In that way the two bacitracin A molecules link two subtilisin molecules together to form a dimer. Despite this common feature we found some important differences in the conformations of bacitracin A in the three complex structures which were analysed and described in detail in this paper. An examination of the solvent structure of the complexes shows water molecules in the region around the bacitracin A molecules are not conserved and play a different role in the stabilization of the bacitracin A conformation.

Anti-Bacterial Agents↗
PubMed1996

Crystal structures of the alkaline proteases savinase and esperase from Bacillus lentus.

Savinase and Esperase (EC. 3.4.21.14) are secreted by the alkalophilic bacterium Bacillus lentus and are representatives of that subgroup of subtilisin enzymes with maximum stability in the range of pH 7 to 10 and high activity in the range of pH 8 to 12. The crystal structures of native Savinase and diisopropyl fluorophosphate (DFP) inhibited Esperase have been refined using X-ray data to 1.4 angstroms and 1.8 angstroms resolution respectively collected with synchrotron radiation. The structures were refined to R-factors (=(Sigma//Fo/-/Fc//)/(Sigma/Fo/)) of 16.4% for Esperase and 17.3% for Savinase. The sequence identity between the two enzymes is 66%. The structures are very similar to those of other Bacillus subtilisins. There are two calcium ions in each, equivalent to the strong and the weak sites in subtilisins Carlsberg and BPN'. The structures show novel features which can to some extent be related to their stability and activity. The large number of salt bridges in Esperase and Savinase is likely to contribute to the high thermal stability. Non-conservative substitutions and deletions in the hydrophobic binding pocket S1 as well as the more hydrophobic character of the substrate binding region probably contribute to the alkaline activity profile of the enzymes. Towards the end of the binding site there is an extra proline, Pro131, in Savinase near proline 129, forming a cluster that provides extra active-site rigidity compared with other subtilisins. On the other side of the active site of Esperase and Savinase, the tyrosine found in most other subtilisins is replaced by leucine and valine respectively. The tyrosine potentially interacts with substrate residue P6. At high pH, the negatively charged deprotonated tyrosine could interact unfavorably with the substrate, a possibility that is overcome by substitution with a neutral residue. This is probably one explanation for the shift of the activity profile of Esperase and Savinase to more alkaline pH.

Alkalies↗
PubMed1995

Purification, crystallization and preliminary X-ray crystallographic analysis of lactoperoxidase from buffalo milk.

The lactoperoxidase was prepared from buffalo milk and purified using CM-Sephadex C-50 and Sephadex G-100. The activity of the enzyme was measured using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt as a chromogenic substrate at pH 6.0. The purified protein was crystallized from 0.01 M sodium phosphate buffer (pH 8.0) with 10%(v/v) ethanol by the sitting-drop vapour-diffusion method. The green-coloured plate-like crystals are orthorhombic in space group P2(1)2(1)2(1) with unit-cell dimensions a = 116.9, b = 103.2 and c = 62.3 A. The asymmetric unit contains one molecule with a solvent content of 52%. The crystals were stable in the X-ray beam and diffract beyond 3.2 A. The native data to 3.5 A have been collected and the structure determination is in progress.

Journal Article↗
PubMed1995

Crystallization and preliminary diffraction studies of the structural domain E of Thermus flavus 5S rRNA.

The ribosomal 5S RNA is an essential constituent of the large ribosomal subunit. To overcome the difficulties of crystallizing large RNA molecules such as 5S rRNAs, we decided to divide the 5S rRNA in five domains A through E to determine their structure. Recently we determined the crystal structural of the helical domain A. Here we report the crystallization of the chemically synthesized domain E of the Thermus flavus 5S rRNA. The crystal form is trigonal with unit cell dimensions: a = b = 42.80 A and c = 162.20 A. Diffraction-data to 2.8 A have been recorded and the structure solution is currently underway by means of MIR and MAD techniques.

Base Sequence↗
PubMed1995

Crystal structure of catalase HPII from Escherichia coli.

BACKGROUND: Catalase is a ubiquitous enzyme present in both the prokaryotic and eukaryotic cells of aerobic organisms. It serves, in part, to protect the cell from the toxic effects of small peroxides. Escherichia coli produces two catalases, HPI and HPII, that are quite distinct from other catalases in physical structure and catalytic properties. HPII, studied in this work, is encoded by the katE gene, and has been characterized as an oligomeric, monofunctional catalase containing one cis-heme d prosthetic group per subunit of 753 residues. RESULTS: The crystal structure of catalase HPII from E. coli has been determined to 2.8 A resolution. The asymmetric unit of the crystal contains a whole molecule, which is a tetramer with accurate 222 point group symmetry. In the model built, that includes residues 27-753 and one heme group per monomer, strict non-crystallographic symmetry has been maintained. The crystallographic agreement R-factor is 20.1% for 58,477 reflections in the resolution shell 8.0-2.8 A. CONCLUSIONS: Despite differences in size and chemical properties, which were suggestive of a unique catalase, the deduced structure of HPII is related to the structure of catalase from Penicillium vitale, whose sequence is not yet known. In particular, both molecules have an additional C-terminal domain that is absent in the bovine catalase. This extra domain contains a Rossmann fold but no bound nucleotides have been detected, and its physiological role is unknown. In HPII, the heme group is modified to a heme d and inverted with respect to the orientation determined in all previously reported heme catalases. HPII is the largest catalase for which the structure has been determined to almost atomic resolution.

Amino Acid Sequence↗