Biomedical subjects

C Betzel

Publications and source records attributed to C Betzel.

At least 37 records · Page 2Linked to original sources

PubMed2001

Mercury induced modifications in the stereochemistry of the active site through Cys-73 in a serine protease--crystal structure of the complex of a partially modified proteinase K with mercury at 1.8 A resolution.

Proteinese K (PK) isolated from Tritirachium album Limber was crystallized with HgCl2 in excess, under microgravity conditions. The intensity data were collected at 4 degrees C to 1.8 A resolution and the final R-factor after refinement for all the reflections was 0.164. Mercury has been found at two sites with partial occupancies (0.4 and 0.6) which are at distances of 2.48 A and 2.58 A respectively from Cys-73 Sgamma. The Cys-73 in the enzyme structure is located close to the active site residue, His-69. This region is completely buried and is not accessible to the solvent. It is rather tightly packed. Therefore, the binding of mercury distorts the stereochemistry of the neighbouring residues including those belonging to the catalytic triad. As a result of this, the Ogamma of Ser-224 is displaced by 0.6 A which causes the inactivation of proteinase K by increasing the H-bond distance to 3.7 A between Ser-224 Ogamma and His-69 Nepsilon2.

Amino Acids↗

PubMed2000

Structure of free Thermus flavus 5 S rRNA at 1.3 nm resolution from synchrotron X-ray solution scattering.

The shape of free Thermus flavus 5 S rRNA in solution at 1.3 nm resolution is restored from synchrotron x-ray scattering data using an ab initio simulated annealing algorithm. The free 5 S rRNA is a bent elongated molecule displaying a compact central region and two projecting arms, similar to those of the tRNA. The atomic models of the 5 S rRNA domains A-D-E and B-C in the form of elongated helices can be well accommodated within the shape, yielding a tentative model of the structure of the free 5 S rRNA in solution. Its comparison with the recent protein-RNA map in the ribosome (Svergun, D. I., and Nierhaus, K. H. (2000) J. Biol. Chem. 275, 14432-14439) indicates that the 5 S rRNA becomes essentially more compact upon complex formation with specific ribosomal proteins. A conceivable conformational change involves rotation of the B-C domain toward the A-D-E domain. The model of free 5 S rRNA displays no interactions between domains E and C, but such interactions are possible in the bound molecule.

Algorithms↗

PubMed2000

Spectroscopic investigation of calcium binding sites in the neurotoxin vipoxin and its components-relation with the X-ray structure.

Vipoxin is a neurotoxin from the venom of Vipera ammodytes meridionalis, the most toxic snake in Europe. It is a unique complex of a toxic phospholipase A2 (PLA2) and a non-toxic PLA2-like protein inhibitor (Inh) which probably evolved from the enzyme and reduces its activity and toxicity. The enzymatic activity of Vipoxin is Ca2+-dependent and the interaction of this metal ion with the neurotoxic complex and its separated components was investigated using the fluorescent probe ANS. Vipoxin binds two calcium ions, one per each subunit. The X-ray model of the Ca2+-free neurotoxin shows that the potential metal-binding sites require minor structural changes to bind calcium. The dissociation constants K(2+)Ca of the calcium complexes of Vipoxin and its components, PLA2 and Inh, were determined to be 16, 10 and 9 mM, respectively. The affinity for calcium of Vipoxin is reduced in comparison to those of PLA2 and Inh. The X-ray model shows that the potential Ca2+-binding sites in the two components are partially 'shielded' in the complex. The affinity of the neurotoxin to Sr2+ and Ba2+ is lower and the respective K(2+)Ca are 20 and 30 mM. The saturation of Ca2+-binding sites increased the melting point Tm of Vipoxin by 11 degrees C and the activation energy for the thermal deactivation of the excited tryptophans Ea by 11 kJ mol(-1) x Ca2+ is important not only for the enzymatic activity of Vipoxin but also for its thermostability.

Animals↗

PubMed2000

Crystallization of engineered Thermus flavus 5S rRNA under earth and microgravity conditions.

Thermus flavus 5S rRNA with a molecular weight of about 40 kDa was modified at the 5' and 3' ends. Crystals were obtained under earth and microgravity conditions. The best crystals were obtained during NASA space mission STS 94. For the first time, it was possible to collect a complete data set from 5S rRNA crystals to 7.8 A resolution and to assign the space group as R32, with unit-cell parameters a = b = 110.3, c = 387.6 A, alpha = beta = 90, gamma = 120 degrees.

Base Sequence↗

PubMed2000

Atomic structure of the Serratia marcescens endonuclease at 1.1 A resolution and the enzyme reaction mechanism.

The three-dimensional crystal structure of Serratia marcescens endonuclease has been refined at 1.1 A resolution to an R factor of 12.9% and an R(free) of 15.6% with the use of anisotropic temperature factors. The model contains 3694 non-H atoms, 715 water molecules, four sulfate ions and two Mg(2+)-binding sites at the active sites of the homodimeric protein. It is shown that the magnesium ion linked to the active-site Asn119 of each monomer is surrounded by five water molecules and shows an octahedral coordination geometry. The temperature factors for the bound Mg(2+) ions in the A and B subunits are 7.08 and 4.60 A(2), respectively, and the average temperature factors for the surrounding water molecules are 12.13 and 10.3 A(2), respectively. In comparison with earlier structures, alternative side-chain conformations are defined for 51 residues of the dimer, including the essential active-site residue Arg57. A plausible mechanism of enzyme function is proposed based on the high-resolution S. marcescens nuclease structure, the functional characteristics of the natural and mutational forms of the enzyme and consideration of its structural analogy with homing endo-nuclease I-PpoI.

Binding Sites↗

PubMed2000

Structure of the bifunctional inhibitor of trypsin and alpha-amylase from ragi seeds at 2.2 A resolution.

The crystal structure of a bifunctional inhibitor of alpha-amylase and trypsin (RATI) from ragi seeds (Indian finger millet, Eleusine coracana Gaertneri) has been determined by X-ray diffraction at 2.2 A resolution. The inhibitor consists of 122 amino acids, with five disulfide bridges, and belongs to the plant alpha-amylase/trypsin inhibitor family. The crystals were grown by the microdialysis method using ammonium sulfate as a precipitating agent. The structure was determined by the molecular-replacement method using as models the structures of Corn Hageman factor inhibitor (CHFI) and of RATI at 2.9 A resolution determined previously. It has been refined to an R factor of 21.9%. The structure shows an r.m.s. deviation for C(alpha) atoms of 2.0 A compared with its own NMR structure, whereas the corresponding value compared with CHFI is found to be 1.4 A. The r.m.s. difference for C(alpha) atoms when compared with the same protein in the structure of the complex with alpha-amylase is 0.7 A. The conformations of trypsin-binding loop and the alpha-amylase-binding N-terminal region were also found to be similar in the crystal structures of native RATI and its complex with alpha-amylase. These regions differed considerably in the NMR structure.

Amino Acid Sequence↗

PubMed1999

Modulation of phospholipase A2 activity generated by molecular evolution.

Snake venom oligomeric neurotoxins offer several unique examples of modulation of phospholipase A2 (PLA2) activity generated by molecular evolution. This phenomenon was found in evolutionary younger snakes and is probably common for representatives of the genus Vipera. At present, the best-studied example is the heterodimeric neurotoxin vipoxin from the venom of the southeast European snake Vipera ammodytes meridionalis. It is a complex between a basic strongly toxic PLA2 and an acidic and catalytically inactive PLA2-like component (Inh). This is the first reported example of a high degree of structural homology (62%) between an enzyme and its natural protein inhibitor. The inhibitor is a product of the divergent evolution of the unstable PLA2 in order to stabilize it and to preserve the pharmacological activity/toxicity for a long time. Inh reduces both the catalytic activity and toxicity of PLA2. Vipoxin also illustrates evolution of the catalytic into a inhibitory function. Vipoxin analogues have been found in the venom of viperid snakes inhabiting diverse regions of the world. An attempt is made to explain modulation of the toxic function by the three-dimensional structure of vipoxin.

Amino Acid Sequence↗

PubMed1999

Crystal structure of mistletoe lectin I from Viscum album.

The crystal structure of the ribosome-inactivating protein (RIP) mistletoe lectin I (ML-I) from Viscum album has been solved by molecular replacement techniques. The structure has been refined to a crystallographic R-factor of 24.5% using X-ray diffraction data to 2.8 A resolution. The heterodimeric 63-kDa protein consists of a toxic A subunit which exhibits RNA-glycosidase activity and a galactose-specific lectin B subunit. The overall protein fold is similar to that of ricin from Ricinus communis; however, unlike ricin, ML-I is already medically applied as a component of a commercially available misteltoe extract with immunostimulating potency and for the treatment of human cancer. The three-dimensional structure reported here revealed structural details of this pharmaceutically important protein. The comparison to the structure of ricin gives more insights into the functional mechanism of this protein, provides structural details for further protein engineering studies, and may lead to the development of more effective therapeutic RIPs.

Abrin↗

PubMed1999

Structure of catalase-A from Saccharomyces cerevisiae.

The structure of the peroxisomal catalase A from the budding yeast Saccharomyces cerevisiae, with 515 residues per subunit, has been determined and refined to 2.4 A resolution. The crystallographic agreement factors R and Rfree are 15.4% and 19.8%, respectively. A tetramer with accurate 222-molecular symmetry is located in the asymmetric unit of the crystal. The conformation of the central core of catalase A, about 300 residues, remains similar to the structure of catalases from distantly related organisms. In contrast, catalase A lacks a carboxy-terminal domain equivalent to that found in catalase from Penicillium vitalae, the only other fungal catalase structure available. Structural peculiarities related with the heme and NADP(H) binding pockets can be correlated with biochemical characteristics of the catalase A enzyme. The network of molecular cavities and channels, filled with solvent molecules, supports the existence of one major substrate entry and at least two possible alternative pathways to the heme active site. The structure of the variant protein Val111Ala, also determined by X-ray crystallography at 2.8 A resolution, shows a few, well-localized, differences with respect to the wild-type enzyme. These differences, that include the widening of the entry channel in its narrowest point, provide an explanation for both the increased peroxidatic activity and the reduced catalatic activity of this mutant.

Alanine↗

PubMed1999

Spectroscopic investigation of phenolic groups ionization in the vipoxin neurotoxic phospholipase A2: comparison with the X-ray structure in the region of the tyrosyl residues.

The neurotoxin vipoxin is the major lethal component of the venom of Vipera ammodites meridionalis, the most toxic snake in Europe. It is a complex between a toxic phospholipase A2 (PLA2) and a non-toxic protein inhibitor (Inh). Tyrosyl residues are involved in the catalytic site (Tyr 52 and 73) and in the substrate binding (Tyr 22). Spectroscopic studies demonstrated differences in the ionization behavior of the various phenolic hydroxyl groups in the toxic PLA2. The tyrosyl side chains of the enzyme can be classified into three groups: (a) three phenolic hydroxyls are accessible to the solvent and titrate normally, with a pKeff = 10.45; (b) three residues are partially 'buried' and participate in hydrogen bonds with neighboring functional groups. They titrate anomalously with a pKeff = 12.17; (c) two tyrosines with a pKeff = 13.23 are deeply 'buried' in the hydrophobic interior of PLA2. They became accessible to the titrating agent only after alkaline denaturation of the protein molecule. The spectroscopic data are related to the X-ray structure of the vipoxin PLA2. The refined model was investigated in the region of the tyrosyl side chains. The accessible surface area of each tyrosyl residue and each phenolic hydroxyl group was calculated. A good correlation between the spectrophotometric and the crystallographic data was observed. The ionization behavior of the phenolic groups is explained by peculiarities of the protein three-dimensional structure and the participation of tyrosines in the catalytic site hydrogen bond network. Attempts are made to assign the calculated pKeff values to individual residues. The high degree of 'exposure' on the protein surface of Tyr 22 and 75 is probably important for their function as parts of the substrate binding and pharmacological sites.

Animals↗

PubMed1999

Crystallization and preliminary X-ray diffraction studies of the neurotoxic, heterodimeric phospholipase A2 from the Taiwan viper (Vipera russelli formosensis).

The 28 kDa heterodimeric complex from Taiwan viper (F4/F7 complex) is composed of a neurotoxic phospholipase A2 (F4) and a non-toxic PLA2-like component (F7). Despite a high sequence identity (65%), the biological and pharmacological activities of F4 and F7 are contrasting. The complex is a structural analogue of Vipoxin found in the venom of the Bulgarian viper Vipera ammodites meridionalis. It is unclear how and why such varied bioactivities are expressed in these similar components. The F4/F7 complex has been crystallized using hanging-drop vapour diffusion and macroseeding techniques. The space group is monoclinic P21 with unit-cell dimensions a = 74.92, b = 85.13, c = 78.16 A and beta = 95.12 degrees. X-ray intensity data to 2.0 A resolution have been collected at 120 K and the structure has been solved using the molecular-replacement method. There are four F4/F7 complex molecules in the asymmetric unit, which do not exhibit any local point-group symmetry.

Animals↗

PubMed1999

Isolation, purification, crystallization and preliminary X-ray analysis of beta 1-bungarotoxin from Bungarus caeruleus (Indian common krait).

Beta-Bungarotoxin is a heterodimeric neurotoxin consisting of a phospholipase A2 subunit linked by a disulfide bond to a K+ channel binding subunit, which is a member of of the Kunitz protease-inhibitor subfamily. Purified beta1-bungarotoxin was crystallized using microdialysis techniques. The rectangular-shaped crystals are orthorhombic, space group C2221, with unit-cell dimensions a = 80.7, b = 82.5, c = 56.9 A. The crystals have a calculated Vm of 2.35 A3 Da-1 for one molecule in the asymmetric unit. This corresponds to a solvent content of 48%. X-ray intensity data were collected to 2.6 A resolution. The data set is 97.4% complete.

Animals↗

PubMed1998

Crystal structure of subtilisin DY, a random mutant of subtilisin Carlsberg.

The crystal structure of subtilisin DY inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone has been solved by molecular replacement with subtilisin Carlsberg as the starting model. The model has been refined to a crystallographic R factor (= sigma absolute value [(absolute value Fo) - (absolute value Fc)] / sigma (absolute value of Fo) of 15.1% using X-ray diffraction data to 0.175 nm resolution. Subtilisin DY is an alkaline proteinase from the X-irradiated Japanese strain DY of Bacillus licheniformis, which normally produces subtilisin Carlsberg. It has very similar properties to subtilisin Carlsberg, with a slightly enhanced resistance to heat and guanidine hydrochloride-induced denaturation, in spite of the fact that the sequences of the two enzymes differ in 31 positions out of 274 residues. The close similarity in overall three-dimensional structure of subtilisins DY and Carlsberg and also their physicochemical properties, such as activity and stability, shows that nature aided by X-irradiation for rapid 'evolution' is able to accommodate considerable changes in sequence without substantial changes in property.

Amino Acid Sequence↗

PubMed1998

Crystal structure of a complex formed between proteolytically-generated lactoferrin fragment and proteinase K.

Lactoferrin is an iron binding glycoprotein with a molecular weight of 80 kDa. The molecule is divided into two lobes representing the N-terminal and C-terminal halves of the polypeptide chain, each containing an iron binding site. The serine proteinases such as trypsin, chymotrypsin, and pepsin hydrolyze lactoferrin into two unequal halves while proteinase K divides this protein into two equal halves. In the first step of hydrolysis by proteinase K, the C- and N-lobes, each having a molecular weight of approximately 40 kDa, are generated. In the next step, the lobes are further hydrolyzed into small molecular weight peptides. The proteinase K isolated from the hydrolyzed product does not show enzymatic activity suggesting that the enzyme is inhibited. Furthermore, the hydrolysis experiments on N-lobe and C-lobe showed that the inhibitory fragment came from the C-lobe. The purified lactoferrin fragment was found to be a decapeptide with an amino acid sequence of H2N-Val-Ala-Gln-Gly-Ala-Ala-Gly-Leu-Ala-COOH. The complex formed between proteinase K and lactoferrin fragment was crystallized by microdialysis. The crystals belonged to the monoclinic space group P2(1) with cell dimensions a = 44.4 A, b = 38.6 A, c = 79.2 A, beta = 105.8 degrees and Z = 2. The crystal structure has been determined at 2.4 A resolution. It has been refined to an R factor of 0.163 for 9044 reflections. The Lf-fragment forms several intermolecular interactions with proteinase K. The Ser-224 Ogamma and His-57 N epsilon2 move away to a distance of 3.68 A in the complex. In the crystal structure, Gln-3I (I indicates inhibitor i.e., lactoferrin fragment) is involved in a direct intermolecular interaction with a symmetry related proteinase K molecule through a strong hydrogen bond with Asp-254. The mode of intermolecular interactions in the complex conformational features of the enzyme and placement of the fragment with respect to the enzyme resemble with the molecular complex of proteinase K with its natural inhibitor PKI3 from wheat.

Animals↗

PubMed1998

Crystallization and preliminary X-ray crystallographic analysis of the EGF receptor ectodomain.

Crystallization of the hydrophilic ectodomain of the epidermal growth factor (EGF) receptor has been accomplished in the presence of the ligand EGF. Two different crystal forms have been obtained, one of which was suitable for X-ray analysis. The space group of this form has been assigned to P21212 with unit-cell dimensions of a = 207.4, b = 113.3 and c = 120.4 A. A native data set has been recorded and a heavy-atom search is currently under way. Diffraction from these crystals, however, is limited to low resolution and extensive trials to improve crystal quality further have all failed. To analyse the molecular shape and aggregation of the receptor protein in solution, small-angle X-ray diffraction and dynamic light-scattering techniques have been applied. Synchrotron radiation in combination with cryo-techniques is essential for data collection because of the high solvent content and radiation sensitivity.

Animals↗

PubMed1998

Primary structure and molecular modeling of mistletoe lectin I from Viscum album.

The first three-dimensional structure of the ribosome inactivating protein mistletoe lectin I (ML-I) from Viscum album has been modeled on the basis of the X-ray structure of castor bean ricin from Ricinus communis. The relative high sequence homology and conserved secondary structure enabled accurate modeling. The 196 sequence changes between ML-I and ricin could be accomodated with only little pertubation in the main chain folding. A close comparison of the primary structures of ML-I and ricin is given and the effects of the sequence changes are elucidated on the basis of the modeled three-dimensional structure. Differences have been identified in the vicinity of the active site, in the high affinity galactose binding site and in the interface between the A and B chains, which might account for the reduced cytotoxicity of ML-I.

Amino Acid Sequence↗

PubMed1998

Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop.

The synthetic RNA fragment 5'-CUGGGCGG(GCGA)CCGCCUGG (nucleotides in parentheses indicate the loop region) corresponds to the natural sequence of domain E from nucleotides 79-97 of the Thermus flavus 5S rRNA including a hairpin loop. The RNA structure determined at 3.0 A and refined to an R-value of 24.1% also represents the first X-ray structure GNRA tetraloop. The loop is in distinctly different conformation from other GNRA tetraloops analyzed by NMR. The conformation of the two molecules in the asymmetric unit is influenced and stabilized by specific intermolecular contacts. The structural features presented here give evidence for the ability of RNA molecules to adapt to specific environments.

Base Sequence↗

PubMed1998

13C NMR, X-ray, and differential scanning calorimetry investigations of truncated BPTI (aprotinin) analogues.

Truncated BPTI missing residues 1 and 2 is investigated together with variants thereof (Lys-15, Arg-17, and Arg-42 are replaced by other residues in various combinations). A comparison of the X-ray structure of BPTI with that of 3-58BPTI(K15R,R17A,R42S) shows only minor variations for the backbone, but the lack of salt bridge between the terminals and the lack of two N-terminal residues provide a structure open at one end. Comparisons of amide exchange rates show a dramatic increase for the most slowly exchanging NH protons of 3-58BPTI and the analogues thereof, as compared to those of the wild-type despite only small differences in the structures. The amide exchange rates for truncated analogues increase with decreasing TTEP (temperature top endothermic peak) values. On the basis of the known structural changes comparisons to 13C chemical shifts are made. 13C chemical shifts are assigned using the D-isotope and HMBC techniques. Excellent resolution is obtained in these 1D natural abundance spectra. 13C NMR chemical shifts are shown to be able to gauge structural changes. A comparison of 13C chemical shifts of WT BPTI (aprotinin) and 3-58BPTI reveals effects caused by (i) the removal of the salt bridge of the terminii, (ii) the charge of the N-terminus, and (iii) the increased mobility of the side chain of Tyr-23. Small effects are also seen due to a conformational change of the aromatic ring of Phe-4. Ring current shifts at 13C chemical shifts are calculated. The difference in the calculated ring current effects are small comparing the wild-type with 3-58BPTI(K15R,R17A,R42S) provided the structures are relaxed. Protein unfolding as a function of pH and temperature is studied by DSC. Unfolding occurs at lower temperature with N-terminally truncated analogues, and the maximum is shifted toward higher pH.

Amino Acid Substitution↗