Biomedical subjects

C Frömmel

Publications and source records attributed to C Frömmel.

14 recordsLinked to original sources

PubMed1990

Prediction of prolyl residues in cis-conformation in protein structures on the basis of the amino acid sequence.

In proteins most peptide bonds are in trans-conformation: the torsion angle omega = 180 degrees. Only few show cis-conformation in known protein structures (omega = 0 degrees). Most of them are prolyl residues. About 6% of about 4000 prolyl residues are in cis-conformation. Between trans- and cis-prolyl residues significant differences are observed in the surrounding sequences. E.g. there are large amounts of aromatic residues N-terminally in case of cis-prolyl residues, but in the case of trans-prolyl residues more aromatic amino acids occur C-terminally. But in all cases there are only complex patterns which are indicative of cis- and trans-conformation, respectively. Considering the neighbours (+/- 6 residues) of prolyl residues and their physicochemical properties we find 6 different patterns which allow one to assign correctly about 75% of known cis-structured prolyl residues, whereby no false positive one is predicted.

Crystallography↗

PubMed1990

Crystal structure of thermitase at 1.4 A resolution.

The crystal structure of thermitase, a subtilisin-type serine proteinase from Thermoactinomyces vulgaris, was determined by X-ray diffraction at 1.4 A resolution. The structure was solved by a combination of molecular and isomorphous replacement. The starting model was that of subtilisin BPN' from the Protein Data Bank, determined at 2.5 A resolution. The high-resolution refinement was based on data collected using synchrotron radiation with a Fuji image plate as detector. The model of thermitase refined to a conventional R factor of 14.9% and contains 1997 protein atoms, 182 water molecules and two Ca ions. The tertiary structure of thermitase is similar to that of the other subtilisins although there are some significant differences in detail. Comparison with subtilisin BPN' revealed two major structural differences. The N-terminal region in thermitase, which is absent in subtilisin BPN', forms a number of contacts with the tight Ca2+ binding site and indeed provides the very tight binding of the Ca ion. In thermitase the loop of residues 60 to 65 forms an additional (10) beta-strand of the central beta-sheet and the second Ca2+ binding site that has no equivalent in the subtilisin BPN' structure. The observed differences in the Ca2+ binding and the increased number of ionic and aromatic interactions in thermitase are likely sources of the enhanced stability of thermitase.

Amino Acid Sequence↗

PubMed1990

Mapping mutations in influenza A virus resistant to norakin.

To elucidate the mode of action of norakin against influenza A virus we sequenced the hemagglutinin gene of 11 norakin-resistant mutants. Resistance was coupled with 1-3 amino acid exchanges. The majority of mutations was localized in the HA2 polypeptide and was mostly associated with changes in charge or polarity of the amino acids. The amino acid substitutions are discussed in the context of the 3D structure of X31 hemagglutinin considered to be representative of the influenza hemagglutinins. Most of the mutations appear to destabilize the pH 7.0 structure by distorting or destroying hydrogen bonds as well as salt-bridges which are responsible for intra- and intersubunit contacts, while others destabilize the location of the fusion peptide, facilitating conformational changes in the presence of the inhibitor.

Amino Acids↗

PubMed1990

[Crystal structure of thermitase and stability of subtilisins].

Crystal structure of thermitase, a serine proteinase from Thermoactinomyces vulgaris, has been determined by X-ray diffraction at 1.4 A resolution. The atomic model of thermitase refined to an R-factor of 0.149 contains 1997 protein atoms, 182 water molecules and 2 Ca2+ ions. The tertiary structure of thermitase is similar to that of subtilisin BPN'. The greatest variations are connected with insertions and deletions in the amino acid sequence, which are located on the surface of the molecule. Higher thermostability of thermitase can be explained in terms of the three-dimensional structure. The Ca2+ ions, bound to the protein molecule, as well as the ionic and hydrophobic interactions are supposed to give the main contribution to the stabilization of the structure.

Amino Acid Sequence↗

PubMed1989

Crystal structure of thermitase from Thermoactinomyces vulgaris at 2.2 A resolution.

The crystal structure of thermitase from Thermoactinomyces vulgaris has been determined by x-ray diffraction at 2.2 A resolution. The structure was solved by a combination of single isomorphous replacement and molecular replacement methods. The structure was refined to a conventional R factor of 0.24 using restrained least square procedures CORELS and PROLSQ. The tertiary structure of thermitase is similar to that of subtilsin BPN'. The greatest differences between these structures are related to the insertions and deletions in the sequence.

Actinomycetales↗

PubMed1989

Thermitase, a thermostable subtilisin: comparison of predicted and experimental structures and the molecular cause of thermostability.

The subtilisin family of proteases has four members of known sequence and structure: subtilisin Carlsberg, subtilisin novo, proteinase K, and thermitase. Using thermitase as a test case, we ask two questions. How good are methods for model building a three-dimensional structure of a protein based on sequence homology to a known structure? And what are the molecular causes of thermostability? First, we compare predicted models of thermitase, refined by energy minimization and varied by molecular dynamics, with the preliminary crystal structure. The predictions work best in the conserved structural core and less well in seven loop regions involving insertions and deletions relative to subtilisin. Here, variation of loop regions by molecular dynamics simulation in vacuo followed by energy minimization does not improve the prediction since we find no correlation between in vacuo energy and correctness of structure when comparing local energy minima. Second, in order to identify the molecular cause of thermostability we confront hypotheses derived by calculation of the details of interatomic interactions and estimates of hydrophobic interactions with inactivation experiments. As a result, we can exclude salt bridges and hydrophobic interactions as main causes of thermostability. Based on a combination of theoretical and experimental evidence, the unusually tight binding of calcium by thermitase emerges as the most likely single influence responsible for its increased thermostability.

Actinomycetales↗

PubMed1989

Polarity as a criterion in protein design.

Hypothetical proteins can be tested computationally by determining whether or not the designed sequence-structure pair has the characteristics of a typical globular protein. We have developed such a test by deriving quantities with approximately constant value for all globular proteins, based on empirical analysis of the exposed and buried surfaces of 128 structurally known proteins. The characteristic quantities that best appear to segregate badly designed or deliberately misfolded proteins from their properly folded natural relatives are the polar fraction of side chains on the protein surface and, independently, in the protein interior. Three of the seven hypothetical structures tested here can be rejected as having too many polar side-chain groups in the interior or too few on the protein surface. In addition, a recently designed nutritional protein is identified as being very much unlike globular proteins. These database-derived characteristic quantities are useful in screening designed proteins prior to experiment and may be useful in screening experimentally determined (X-ray, NMR) protein structures for possible errors.

Electric Conductivity↗

PubMed1988

Use of the averaged mutation rate in pieces of protein sequences to predict the location of antigenic determinants.

Antigenic determinants in proteins show properties, which can be used to their prediction: high degree of accessibility, mobility, polarity etc. Furthermore, there is a remarkable correlation between the location of antigenic determinants and regions with high frequency of accepted mutations during the evolution. Consequently, it is possible to predict successfully the location of antigenic epitopes in proteins using the averaged mutation rate of protein sequence pieces.

Amino Acid Sequence↗

PubMed1988

Oxidation of a methionine residue in subtilisin-type proteinases by the hydrogen peroxide/borate system--an active site-directed reaction.

Subtilisin-type proteinases (thermitase, subtilisin Carlsberg, alkaline proteinase ZIMET 10911, proteinase K) are partially inactivated by hydrogen peroxide in the alkaline pH range only in the presence of boric acid or phenylboronic acid. A model is presented to describe the inactivation mechanism. Both boric acid and perboric acid existing in equilibrium in the presence of hydrogen peroxide bind competitively at the active site of the enzyme. The inactivation, which is known to be caused by sulfoxide formation from the methionine residue in the active site (Stauffer, C.E. and Etson, D. (1969) J. Biol. Chem. 244, 5333-5338), is due to the enzyme-bound perboric acid species. The dissociation constants for the boric acid-thermitase and perboric acid-thermitase complexes are 36 +/- 7 and 4 +/- 1 mM, respectively. The first-order rate constant of inactivation is k = 0.63 +/- 0.14 min-1. The same mechanism of inactivation holds true for phenylboronic acid in alkaline hydrogen peroxide solutions.

Bacillus↗

PubMed1987

[Long-term stability of enzymes in solution].

Two proteases (thermitase, a thermostable serine protease from Thermoactinomyces vulgaris and subtilisin Carlsberg) and one non-proteolytic enzyme (urate oxidase from Penicillium spp.) were used for revealing the main influences leading to the inactivation of enzyme preparations during their long-time storage at low temperatures. The temperature dependences (0 degree C-60 degrees C) of inactivation resulted in a linear Arrhenius-plot for each of the three native enzymes as well as in the presence of all stabilizing substances tested. Therefore, a method is available which shortens the time considerably needed for the experiments aimed at the discovery of substances stabilizing enzymes under storage conditions (i.e. long time at low temperatures). Because of the linear Arrhenius-plots potential stabilizers can be tested experimentally at suitable higher temperatures and one can extrapolate on their influence on the enzyme at storage temperatures (0 degree C-10 degrees C). By using this method effective combinations of stabilizers for urate oxidase were found, and possible reasons of their stabilizing influence on the enzyme are discussed.

Cold Temperature↗

PubMed1985

The use of progress curves for the estimation of inactivation rate constants of enzymes.

Progress curve analysis is applied to the estimation of inactivation rate constants. The heat inactivation of inorganic pyrophosphatase from baker's yeast was studied assuming a simple one-substrate mechanism containing two irreversible inactivation steps for the free enzyme and the enzyme-substrate complex, respectively. Four kinetic parameters including Km simultaneously may be detected from one progress curve. The temperature dependences derived from the estimated rate constants for several inactivating temperatures agree well with that obtained from preincubation measurements.

Biometry↗

PubMed1979

[Effect of magnesium ions on the thermostability of inorganic pyrophosphatase from baker's yeast].

The interaction of magnesium ions with inorganic pyrophosphatase from baker's yeast was studied by means of heat denaturation. The heat inactivation of this enzyme is a biphasic process. The velocities in the initial range and in the subsequent slower part of inactivation are diminished with rising Mg2+ concentration in the inactivation assay. A model is proposed which describes this behavior. It is assumed that two enzyme conformations exist in equilibrium whose conversion rates correspond to the inactivation rate in its order of magnitude. The equilibrium is shifted by Mg2+. The two enzyme species differ in their Mg2+ binding behavior as evidenced by differences in the half-saturation constants and the cooperativity of the binding. The same conclusions are drawn from the fluorimetric measurement of denaturation of inorganic pyrophosphatase. Besides, an additional Mg2+ binding site is demonstrable, the saturation of which obviously leads to stabilisation of part of the enzyme structure without protecting it against loss of enzymatic activity. With the same method the labilizing effect of Zn2+ on the structure of the inorganic pyrophosphatase from baker's yeast was studied.

Drug Stability↗

PubMed1978

[Characterization of a protease from Thermoactinomyces vulgaris (thermitase). 1. Purification of thermitase].

The paper deals with the purification of the microbial protease preparation "thermitase" (submerged cultivation of Thermoactinomyces vulgaris; treatment of culture filtrate with ethanol or Na2SO4, vacuum drying of precipitate). The crude substance was purified by column chromatography on Sephadex G-75, DEAE-Cellulose and Sephadex G-50. The proteolytically active fractions were in each case united, freeze dried and tested for protein components and protease activity by gel electrophoresis. After passage of the third column the isolated protease (4.5 fold enrichment in the specific activity) was further characterized. The electropherogram (pH 8.9) presented a protease band moving to the anode which was accompanied by 2 very weak protease bands. Furthermore there could be detected a very active protease band (main component of Thermitase) as well as a side band with lower activity both moving to the cathode. The freeze dried preparation contained 85% protein and 4% carbohydrates (glucose as single monomer component after acid hydrolysis). A molecular weight of 11,000 was determined by chromatography on Sephadex G-75. This value is critically discussed. Hints are given for autolytic processes taking place during the purification procedure.

Micromonosporaceae↗

PubMed1978

[Characterization of a protease from Thermoactinomyces vulgaris (thermitase). 2. Single-step fine purification and protein-chemical characterization].

The fine purification of an alkaline protease (thermitase) from Thermoactinomyces vulgaris by means of isoelectrical focussing in the flat-bed procedure using granulated gel is reported. An Na2SO4-precipitated crude product serves as the starting material. Isoelectrical focussing leads in a single step to a highly purified protein with an uniform N-terminal end group. The enzyme has an IP at 9.0 and a mol. wt. of 37,400; it consists of a polypeptide chain with arginine as the N-terminal, and tyrosine as the C-terminal end group. In addition to an essential serine residue, a SH group could be demonstrated which is hardly accessible in the native enzyme. Furthermore, the influence of different protease inhibitors was studied.

Isoelectric Focusing↗