Otto Epp

The basic trypsin inhibitor of bovine pancreas

The basic pancreatic trypsin inhibitor was first described by Kunitz and Northrop ~t]. I t binds to and inhibits trypsin with a binding constant of approximately t013 (M -I) at alkaline pH [21. I t also inhibits e-cliymotrypsin, kallikrein, and plasmin [31. I t has a molecular weight of 6,500 and consists of 58 amino acids. The amino acid sequence has been determined by Kassell and Laskowski [4] and by Anderer and H6rnle ES] (Fig. 1). I t contains three disulfide bridges and has a large excess of basic residues over acidic amino acids.

Structure analysis and molecular model of the selenoenzyme glutathione peroxidase at 2.8 A resolution.

Abstract The three-dimensional structure of bovine erythrocyte glutathione peroxidase, a tetrameric enzyme containing 4 gram atoms of selenium per mole ( M r = 84,000), has been determined at 2.8 A resolution using the multiple isomorphous replacement method. By correlation calculations in Patterson space the tetramers were shown to exhibit molecular [222] symmetry, proving the monomers to be identical or at least very similar. The monomer consists of a single polypeptide chain of 178 amino acid residues. Its shape is nearly spherical with a radius of r ≈ 19 A . A tentative sequence corresponding to a partially refined model ( R = 0.38) is given. Each subunit is built up from a central core of two parallel and two anti-parallel strands of pleated sheet surrounded by four α-helices. One of the helices runs antiparallel to the neighbouring β-strands giving rise to a βαβ substructure, an architecture that has been found in several other proteins e.g. flavodoxin, thioredoxin, rhodanese and dehydrogenases. A comparison of the glutathione peroxidase subunit structure with thioredoxin-S 2 revealed large regions of structural resemblance. The central four-stranded β structure together with two parallel α-helices resembles nearly 80% of the thioredoxin fold. The active sites of glutathione peroxidase are located in flat depressions on the molecular surface. Probably each active centre is built up by segments from two subunits. The catalytically active selenocysteines were found at the N-terminal ends of long α-helices and are surrounded by an accumulation of aromatic side-chains. A difference Fourier map between oxidized and substrate-reduced glutathione peroxidase as well as heavy-atom binding led to the conclusion that the two-electron redox-cycle involves a reversible transition of the active-site selenium from a selenenic acid (RSeOH) to a seleninic acid (RSeOOH).

Crystallization, crystal structure analysis and preliminary molecular model of the bilin binding protein from the insect Pieris brassicae

The bilin binding protein of the butterfly Pieris brassicae has been prepared, crystallized and its crystal structure determined at high resolution using film and FAST area detector intensity data. The crystallographic asymmetric unit contains a tetramer of identical subunits with a molecular weight of about 90,000. The crystal structure was determined by isomorphous replacement. Use was made of the molecular symmetry to improve phases. A molecular interpretation of the electron density distribution and partial tracing of the polypeptide chain was possible without amino acid sequence information, as the fold is very similar to retinol binding protein. It is characterized by a beta-barrel formed by two orthogonal beta-sheets and an alpha-helix. The bilin pigment seems to be bound within the beta-barrel analogously to retinol in retinol binding protein. The tetramer in the crystal has C2 symmetry and is a dimer of dimers of quasi-equivalent subunits.

Structures of deoxy- and carbonmonoxy-erythrocruorin

Abstract The structures of the deoxy and carbonmonoxy derivatives of the insect haemoglobin erythrocruorin have been determined by difference Fourier syntheses. In deoxyerythrocruorin the iron atom of the haem group was found to be five-coordinated. The carbonmonoxy- versus deoxy- difference map showed that the carbon monoxide molecule is inclined to the haem plane. Slight structural changes could be observed to take place on carbon monoxide binding. These structural changes are probably due to the difference in spin state between deoxy- and carbonmonoxy haemoglobin.

The structure determination of the variable portion of the Bence-Jones protein Au

The structure of a χ-type Bence-Jones protein variable fragment Au has been determined by molecular replacement methods using the known structure of an other Bence-Jones variable fragment Rei (Epp et al., Eur. J. Biochem. 45, 513 (1974)). The crystallographic R factor is 0.31 for about 4000 significantly measured reflections between 6.8 to 2.5 å. The Au protein forms a dimer across a crystallographic two fold axis. The spatial relationship of the two monomers, the conformation of the backbones and of the internal residues is extremely similar to that found in Rei.

Solution of the phase problem in the X-ray diffraction method for proteins with the nuclear magnetic resonance solution structure as initial model: Patterson search and refinement for the α-amylase inhibitor tendamistat

Patterson search calculations using the three-dimensional structure of the alpha-amylase inhibitor from Streptomyces tendae obtained from experimental nuclear magnetic resonance (n.m.r.) data were performed to study the possibility of solving the phase problem in the X-ray diffraction method with protein structures determined by n.m.r. Using all heavy atoms (C, N, O, S) of the residues 5 to 73 in the best n.m.r. structure of the alpha-amylase inhibitor (520 out of the 558 heavy atoms in the complete polypeptide chain), the maximum of the rotation function corresponded to the correct solution obtained by the previous independent determination of the crystal structure. However, additional local maxima, which are not significantly lower than the global maximum, also showed up. Performing the Patterson search with a model containing the backbone atoms and the heavy atoms of only the interior side-chains (399 atoms), which are much better defined by the n.m.r. data, the correct maximum was significantly higher than all other maxima. A translation search for the best orientation of the latter model yielded the correct solution. The energy-restrained crystallographic refinement was performed with this model to an R-factor of 26%. This corresponds approximately to the R-factor calculated for the X-ray crystal structure previously determined using the isomorphous replacement technique, if the residues 1 to 4 and 74 and all localized solvent molecules were removed from this structure. During the refinement the root-mean-square deviation between the two structures decreased from 1.03 A to 0.26 A for the polypeptide backbone and from 1.64 A to 0.73 A for all heavy atoms. There are no major local conformational differences between the two structures, with the single exception of the side-chain of Gln52.

Aufklärung der Molekülstruktur des Insektenhämoglobins

ZusammenfassungDie Tertiärstrukturen des Insektenhämoglobins Erythrocruorin und der Vertebraten-Hämoglobine sind sehr ähnlich. Eine Ausnahme bildet ein Knick in der Helix H.Die Sekundärstrukturen von Erythrocruorin und Myoglobin unterscheiden sich nur in einigen Aminosäuredeletionen, deren Auswirkungen auf die Struktur gering und eng begrenzt sind. Die Deletionen sind fernab der Hämingruppe. Die “benzolische” Umgebung der Hämingruppe ist bemerkenswert [10]. Eine oder zwei bisher invariante Aminosäuren scheinen ausgetauscht zu sein (leu F 4, His E 7 Problem). Unsere weiteren Pläne sind, die Messungen bis 2,5 Å Auflösung fortzuführen, und eine pH-abhängige Strukturänderung, die wir an den Einkristallen entdeckten, im atomaren Bereich zu analysieren.AbstractThe tertiary and secondary structure of the insect hemoglobin Erythrocruorin prove basicly similar to the structure of the vertebrate hemoglobins. Some deletions could be detected in comparsion with myoglobin. The structural changes, however, induced by these deletions, are confined to small parts of the molecule away from the heme group. One or two of the invariant amino acids are exchanged.

The environment of the haem group in erythrocruorin (Chironomus thummi).

Abstract The environment of the haem group of the insect haemoglobin erythrocruorin is described as found from a 2.8 A resolution Fourier synthesis. It proved basically similar to the haem group environment of the mammalian haemoglobins. Some interesting amino acid replacements could be detected, the most important being the substitution of the invariant amino acid Leu F4 for an aromatic amino acid.

Kristallstrukturanalyse des Met-Erythrocruorins bei 5,5 Auflsung

Erythrocruorin ist das sauerstoff-tibertragende H~mprotein der Larven der Chironomiden (Chironomus thummi, Zuckmticken). Es ist in der K6rperfltissigkeit gelSst und hat wahrscheinlich, wenigstens in einem bestimmten Sauerstoffdruckbereich, Transportund Speicherfunktion [t~. Eine Bestimmung der Terti~irstruktur dieses Proteins ist insbesondere wegen der Vergleichsmaglichkeit mit den bekannten Terti/irstrukturen der S~iugerhgmproteine Myoglobin [21 und H~imoglobin [3] interessant I4]. Die Reindarstellung yon Erythrocruorin erfordert mehrere F~ilIungen und Chromatographien [5, 6]. Es lassen sich bis zu acht verschiedene Erythrocruorine trennen [7]. Die Kristallisation gelang bisher nur bei einer Bande der DEAE-Cellulosetrennung [51, und zwar in der tiblichen Weise durch Aussalzen in Ammonsulfatpuffer. Wir fanden r6ntgenographisch ein Molekulargewicht yon etwa 16000, einen Weft, der deutlich niedriger als der yon Myoglobin (t7 500) ist. Die Sauerstoff-Affinit~itsmessungen am reinen Erythrocruorin I81 liefern eine Kurve, die sehr /ihnlich der yon Myoglobin ist. Die optische Rotationsdispersion [9~ zeigt einen Helixgehalt yon etwa 50% (also nur ~des Helixgehaltes yon Myoglobin). Tabelle I i s t eine Aufstellung der Elementarzellenkonstanten der bisher erhaltenen Kristallmodifikationen. Die trigonale Modifikation, mit der wir die Kristalistrukturanalyse durchftihrten, kristatlisiert zwischen etwa pH 3,9 und pH 7,5, die orthorhombische bei pH 8,5. Schweratomderivate des Erythrocruorins versuchten wir durch Diffusion in die Kristalle in Ammonsulfatpuffer zu erhalten, jedoch ohne ErIolg. Erst nach einem Austausch des Ammonsulfatpuffers gegen Kalium-Natrium-phosphatpuffer It01 fanden wir auf r6ntgenographischem Wege Schweratomsubstitution. Von den etwa 30 untersuchten Schweratomverbindungen stellten sieh ftinf als besonders giinstig heraus:

The crystallization of protein BL17 from the 50 S ribosomal subunit of Bacillus stearothermophilus.

At present very little is known about the tertiary structure of ribosomal proteins from Escherichiu coli. The shapes of ribosomal proteins have been determined by various techniques, which show some ribosomal proteins to be globular while others are more asymmetric [ 11. More detailed information on the secondary and tertiary structure of these proteins has been obtained by spectroscopic techniques such as circular dichroism (CD) and proton magnetic resonance [2]. The latter experiments, which were on proteins isolated under very mild conditions [3], have shown that many of the E. coli ribosomal proteins have a unique secondary and tertiary structure. The determination of the three-dimensional structure of ribosomal proteins by X-ray diffraction analysis of protein crystals is not yet possible. Despite many attempts at crystallization of intact ribosomal protein crystals suitable for X-ray analysis have not been obtained, although the crystallization of two fragments of proteins L7/12 has been reported [4]. In view of the largely negative results with E. coli proteins we have undertaken the isolation of ribosomal proteins from thermophilic bacteria under mild conditions with a view to their crystallization. Ample evidence exists for the increased resistance of thermophilic proteins against various denaturing conditions [5], thereby increasing the probability of crystallization. From the thermophilic bacteria, the ribosomal proteins of Bacillus stearothermophilus have been