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W Steigemann

Publications and source records attributed to W Steigemann.

18 recordsLinked to original sources
PubMed1992

X-ray crystallographic characterisation of type-2-depleted ascorbate oxidase from zucchini.

The type-2 depleted form of ascorbate oxidase from zucchini has been prepared in crystals and characterised by X-ray crystallography and EPR spectroscopy. The X-ray structure analysis by difference-Fourier techniques and refinement shows that, on average, about 1.3 Cu atoms/ascorbate oxidase monomer are removed. The copper is lost from the trinuclear site whereby the EPR-active type-2 copper is depleted most; type-1 copper is not affected. This observation indicates preferential formation of a 1 Cu-depleted form with the hole equally distributed over all three copper sites. Each of these 1 Cu-depleted species may represent an anti-ferromagnetically coupled copper pair which is EPR-silent and could explain the disappearance of the type-2 EPR signal.

Ascorbate Oxidase↗
PubMed1987

Structural disorder in proteins. A comparison of myoglobin and erythrocruorin.

The refinement of X-ray structural data gives the mean square displacements, (chi 2), at each position in the protein molecule. In order to get information on the significance of such values different refinement methods have been compared. The metmyoglobin structure was determined at 300 K and (chi 2)-values were obtained with the restrained refinement procedure in reciprocal space of Konnert and Hendrickson. A comparison with the results of Frauenfelder et al. was used for an error estimation. The inclusion of surface bound water increases the accuracy of the results but does not change the general picture. For erythrocruorin (CTT3) a refinement was performed in reciprocal space and compared with a refinement in real space performed earlier. The (chi 2)-values obtained from both procedures are similar although the reciprocal space refinement gives results which are physically more reasonable. A comparison of the disorder in myoglobin and erythrocruorin showed that the structural similarity results in a similarity in the disorder. Contacts of molecules in the crystal do not dominate the disorder although they locally influence (chi 2)-values. CTT3 shows large disorder in the heme region in contrast to myoglobin. The differences in the rigidity of the F-helix can be correlated with the oxygen affinities supporting models for O2 binding developed by Frauenfelder et al.

Crystallography↗
PubMed1987

Low temperature X-ray investigation of structural distributions in myoglobin.

The results of X-ray structure analysis of metmyoglobin at 300 K, 185 K, 165 K, 115 K and 80 K are reported. The lattice vectors a and b decrease linearly with temperature while c shows non-linearity above 180 K, indicating some type of phase transition. Cooling does change the myoglobin structure but only within the structural distribution as determined by individual (chi 2)-values at room temperature. Two residues showed significant alternative positions for side-chains at higher temperatures while only one position is occupied at low temperatures. In the case of LEU 61 a jump between different positions of the side-chain reduces the potential barrier for the entrance of the O2 molecule to the heme pocket. The mean square displacements, (chi 2), of the individual residues decrease linearly with temperature in most cases, indicating a parabolic envelope for the potential responsible for motions. A separation of rotational and translational disorder of the entire molecule is discussed. Comparison with Mössbauer spectroscopy indicates that protein dynamics on a time scale faster than 10(-7) s is not simply a harmonic process. Extrapolation of the structural distributions to T = 0 K shows that a large zero point distribution of the myoglobin structure exists, thus proving that there is no absolute energy minimum for one well defined conformation.

Binding Sites↗
PubMed1986

Structure, antigenic determinants of some clinically important insect allergens: chironomid hemoglobins.

Determination of the molecular structure and properties of allergens that elicit severe immediate-type hypersensitivity diseases in humans and a knowledge of the structure of their antibody-binding sites should provide new insight into the pathogenetic mechanisms of allergic diseases. Monomeric and homodimeric hemoglobins (CTT I to X) have been identified as potent allergenic components of Chironomidae, a family of Diptera. Immunologic investigations of peptides of three of these hemoglobins (CTT IV, CTT VI, and CTT VIII) showed that human antibodies of the E and G classes recognize at least two different sites within each molecule. Individual hemoglobin peptides were aligned with homologous regions of chironomid hemoglobin CTT III, whose tertiary structure has been determined by x-ray analysis at a resolution of 1.4 angstroms. The antigenic site CTT IV(91 to 101) showed the following characteristics: (i) seven polar or hydroxylated amino acids, from a total of eleven, occupying predominantly superficial regions; (ii) the property of linkage to other molecules by hydrogen bonds or solvent clusters; and (iii) high thermal mobility factors. In contrast, peptide CTT IV(102 to 108), which does not bind human antibodies, contained no polar amino acids and had low thermal mobility factors. These results support the idea that the antigenicity of clinically relevant proteins is related to regions with a predominance of polar amino acids and with low energy barriers between different conformations, which allow high flexibility, including site-specific adaptation in antibody binding.

Allergens↗
PubMed1985

Structural model of the collagen-like region of C1q comprising the kink region and the fibre-like packing of the six triple helices.

A detailed three-dimensional model of the collagenous part of C1q was derived by model building and computer-aided energy refinement calculations. The proposed structure is based on the collagen-like (-Gly-Xaa-Yaa-) repeating sequence of 78 to 81 residues in the N-terminal regions of the constituent A, B and C chains, on the mode of disulphide linkage between the 18 chains of C1q, and on its electron microscopically derived gross structure. It is demonstrated that the interruptions of the repeating sequence about half-way along the length of the collagenous regions (Gly36-Ile37-Arg38-Thr39 in the A chain and Ala36-Ile37-Hy138 in the C chain) do not lead to a disruption of the triple helical conformation but rather to a bend of about 60 degrees in an otherwise continuous triple helix. These features are consistent with a flexibility comparable with that of regular triple helices and with the observed low proteolytic susceptibility of the kink region. The azimuthal orientation of the kink is defined approximately by ArgA38 being located in the cap of the knee. Because of this extra residue between two glycine residues, a bad contact that would arise between the methyl group of AlaC36 and the peptide carbonyl of IleA37 in a straight triple helix is relaxed. The model features also a cluster of hydrophobic contacts between large hydrophobic side-chains in the interaction edges between the six collagen triple helices aligned with their about 10 nm long N-terminal regions in the fibril-like endpiece of C1q. The azimuthal orientations of the triple helices were derived by energy calculations of side-chain interactions previously applied to fibre-forming collagens. Independently, the same orientations and interaction edges were derived from the azimuthal orientation of the kink and the electron microscopically observed orientations of the triple helical arms that emerge from the endpiece, and which carry the C-terminal globular binding domains. The structural model has a number of implications for the assembly of the first component of complement from C1q and the zymogen complex C1r2C1s2 and possible mechanisms of its activation.

Amino Acid Sequence↗
PubMed1982

Conformational substates in a protein: structure and dynamics of metmyoglobin at 80 K.

The crystal structure of sperm whale metmyoglobin has been determined at 80 K to a resolution of 2A. The overall structure at 80 K is similar to that at 300 K except that the volume is smaller. Refinement of the structure by the method of restrained least squares (current R = 0.175) permits the assignment of isotropic atomic mean-square displacements to all nonhydrogen atoms. Comparison with the values obtained earlier at 250-300 K indicates that the protein at 80 K is more rigid. The average experimentally determined Debye-Waller factor, B, for the protein is 14A2 at 300 K and 5A2 at 80 K. Plots of backbone mean-square displacement vs. temperature show a discontinuity of slope for at least one-third of all residues. This behavior is in good agreement with the temperature dependence of the mean-square displacement of the heme iron as measured by Mössbauer absorption. The magnitudes of the smallest mean-square displacements observed at 80 K indicate that intramolecular motions can be frozen out to a surprisingly large degree. Even at 80 K, however, some atoms in myoglobin still have mean-square displacements greater than 0.1A2, thus providing evidence for conformational substates.

Animals↗
PubMed1975

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

The structure of a pi-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 A. 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.

Amino Acids↗
PubMedDate not supplied

Calculation of the circular dichroism of Chironomus hemoglobin in the light of the quality of its X-ray structure.

For the larval hemoglobin III of the insect Chironomus thummi thummi, the rotational strength of the B (Soret) and Q band is calculated with a monopole/monopole and a dipole/dipole approximation applied to the atomic co-ordinates of the X-ray structures at 2.5 and 1.4 A resolution. In previous calculations using the 2.5 A co-ordinates and with perturbing groups being restricted to aromatic side-chains, the dipole/dipole approximation clearly confirmed the negative sign observed by experiment. The predominant interactions were those of phenylalanine residues. Recalculation with X-ray data refined to the present limits of performance corroborates the negativity of the rotational strength, but now the most important contributions are due to the peptide bonds formerly neglected. Also for the 2.5 A coordinates it is learned that in contrast to earlier expectation the influence of the backbone in this hemoglobin is rather strong. A substantial contribution is further obtained for the perturbation by one of the propionic acid carboxylate groups of the heme. Perspectives and problems of the approach are outlined.

Amino Acids↗