Peter Wilhelm

The molecular size and shape of the extracellular hemoglobin of Nephtys incisa

Abstract The molecular size, shape and conformation of the extracellular hemoglobin of Nephtys incisa were examined using scanning transmission electron microscopy, small-angle X-ray scattering and circular dichroism measurements. In electron micrographs, the overall shape of negatively stained Nephtys hemoglobin was similar to the two-tiered, hexagonal appearance of other annelid extracellular hemoglobins: the vertex-to-vertex diameter of the molecule and its height were 31.6± 1.1 nm and 19.8± 1.2 nm, respectively. An important difference was the presence of an additional subunit, 7.1± 1.1 nm in diameter, in the central cavity of the molecule. The ellipticity of Nephtys hemoglobin over the range 200–250 nm was found to be appreciably more negative than that of other extracellular annelid hemoglobins: the α-helical content of Nephtys oxyhemoglobin was estimated to be 50–60%. The radius of gyration, the maximum diameter and the volume of Nephtys hemoglobin calculated from the small-angle X-ray scattering in solution, at neutral pH, were 10.62 ± 0.15 nm, 28 ± 1 nm, and 6500 nm 3 , respectively. The scattering curves were fitted best by a model consisting of 12 spherical subunits 10.3 nm in diameter, arranged in a hexagonal bilayer with a 13th spherical subunit, 5.6 nm in diameter, situated in the center. The scattering curves of Nephtys hemoglobin were compared with those obtained previously (Pilz, I., Schwarz, E. and Vinogradov, S.N. (1980) Int. J. Biol. Macromol. 2, 279–283) for Lumbricus terrestris and Arenicola marina (Wilhelm, P., Pilz, I. and Vinogradov, S.N. (1980) Int. J. Biol. Macromol. 2, 383–384) hemoglobins.

Applications of FT-IR microscopy with materials analyses

Abstract Some applications of FT-IR microscopy in materials analyses are reported. IR spectra were taken in transmission, using a sample compression cell, or in ATR mode, using an ATR objective for the infrared microscope. Defects and inclusions in polymers and rubbers were chemically characterized. Limitations of the method are discussed, and the importance of sample preparation and proper selection of the infrared spectral technique for intractable samples is emphasized.

Comparison of the structures of the native form of rat liver 5S rRNA and yeast tRNAphe: small-angle and wide-angle X-ray scattering study

The small-angle and wide-angle X-ray scattering of tRNAphe (yeast) and ribosomal 5S RNA (rat liver) in solution have been analysed and compared. tRNAphe in solution is folded into a compact L-shaped structure similar to its structure in crystals. The geometry of the secondary structure of the double helical regions is also equivalent to the A-form in the crystalline state. Despite differences between the molar mosses of 5S rRNA (40 000 g mol−1) and tRNAphe (25 000 g mol−1), and the fact that the 5S rRNA molecule is more anisometric than the tRNAphe molecule, there are many structural similarities. The geometrical parameters of the secondary structure of double helical regions in both RNA molecules are almost identical; the mean rise per base pair is about 0.253–0.28 nm and the mean turn angle is about 32.5–33.5. Identical cross-sectional radii of gyration, Rsq,1 ≈ 1.16 nm and Rsq,2 = 0.92 nm, identical molar mass per unit length, MΔx = 2500 g mol−1 nm−1, and a mean thickness of the molecules D ≈ 1.65 nm suggest a similar, nearly coplanar organization of isolated, double helical arms. Furthermore, there are compact regions in the central parts of both molecules, which are the sites of tertiary interactions in the tRNAphe molecule and are a potential site of tertiary interactions in the SS rRNA molecule for stabilization of the complicated L-shape of the two molecules. Both molecules have a pseudo-twofold axis,w hich may play a role in recognition for binding of specific proteins.

Quaternary structure of immunoglobulin m: A model based on small-angle X-ray scattering data

Abstract This paper presents some data on a human Waldenstrom immunoglobulin M.IgM GAL based on small-angle X-ray scattering data. the IgM GAL had molecular weight 970 000, volume 1760 nm 3 , radius of gyration 12 nm and maximum diameter 37 nm. The conclusions from various model calculations are discussed. A flat, star-shaped model is compatible both with X-ray scattering data and electron micrographs.

Molecular size and shape of the native and reassociated forms of the extracellular haemoglobin of Tubifex tubifex

The extracellular haemoglobin of Tubifex tubifex and the product of its reassociation at neutral pH subsequent to dissociation at alkaline pH, were examined by small-angle X-ray scattering. The following molecular parameters were determined for the native and reassociated molecules, respectively: maximum diameter 30.0±1.0 and 32.0±1.0nm; radius of gyration 10.66±0.15 and 11.07±0.15 nm; molecular weight (3.09±0.15) × 106 and (2.99±0.15) × 106 dalton. Although the scattering curves of the native and reassociated haemoglobin possess similar shapes the distance distribution functions exhibit slight differences in their shape as well as in the position of their maximum. The best fit with the experimental distribution functions was obtained with models consisting of 12 spheres arranged in two hexagonal layers. In the case of the native haemoglobin each of the 12 spheres has a diameter of 9.3 nm while for the reassociated haemoglobin each of the 12 spheres has a diameter of 11.5 nm. The results suggest that although their molecular weights are the same, the reassociated molecule is slightly larger than the native molecule

X-ray scattering of antibodies: the monomeric 8S subunit of human IgM

Abstract 8S monomeric subunit of a human immunoglobulin M was investigated by small-angle X-ray scattering. The following molecular parameters were determined: radius of gyration 5.9 nm, maximum length 21 nm, hydrated volume 410 nm 3 and two radii of gyration of the cross-section: 2.6 and 1.8 nm. A model equivalent in scattering was found and compared with the model for a human IgG also based on small-angle X-ray scattering data. The Cμ2 domain of the IgM obviously has a very loose structure, and the Fab angle of the 8S IgM (90°) is smaller than that of the IgG (134°).

Structure of Annelid Hemoglobins — Small Angle X-Ray Studies

Small angle X-ray scattering is a suitable method to get information on the quaternary structure of extracellular hemoglobins in solution. The method allows to calculate directly from the scattering curve and the distance distribution function (p(r)-function) molecular parameters as molecular weight Mr, radius of gyration R and the maximum distance Dmax. The scattering curve reflects the conformation of the macromolecule in the reciprocal space; the distance distribution function reflects the molecule in the real space and represents the frequency of the distances combining any volume element with any other within the molecule. Models describing the conformation of the hemoglobins in solution can be deduced by comparing their scattering curves and p(r)functions with the experimental ones (1, 2).

Small- and Large-Angle X-Ray Scattering Studies of Counter Ion Influence on tRNA Conformation*

Abstract The determination of cross sectional radii of gyration by small-angle X-ray scattering and comparison of large-angle scattering curves were used to study conformational changes of tRNAs depending on the concentration of mono-and divalent counter ions.

Small-angle X-ray scattering studies of the open and closed conformations of aspartate aminotransferase

SummaryAspartate aminotransferase was investigated by X-ray small-angle scattering. A small difference was found between the “open” (active) and the “closed” (liganded) conformation of the enzyme. The results were compared with X-ray crystallography data.ZusammenfassungAspartat-Aminotransferase wurde mittels Röntgenkleinwinkelstreuung untersucht. Ein kleiner Unterschied zwischen der „offenen“ (aktiven) und der „geschlossenen“ (ligandierten) Konformation wurde gefunden. Die Ergebnisse wurden mit Röntgenkristallstrukturdaten verglichen.

Small-angle X-ray study on the structure of ribulose-1,5-bisphosphate carboxylase-oxygenase from Rhodospirillum rubrum

The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L2, was investigated by small-angle X-ray scattering. In the presence of HCO3-and Mg2+, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm3. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO3-and Mg2+, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L8S8, where inactivation gives rise to substantial changes in configuration.