Ingrid Pilz

Structural and functional domains of cellobiohydrolase I from trichoderma reesei

Limited proteolysis (papain) of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei led to the seperation of two functional domains: a core protein (55 kDa) containing the active site, and a C-terminal glycopeptide (10 kDa) implicated in binding to the insoluble matrix (cellulose). The quaternary structures of the intact CBH I and its core in solution are now compared by small angle X-ray scattering (SAXS) measurements. The molecular parameters derived for the core (Rg=2.09 nm, Dmax=6.5 nm) and for the intact enzyme (Rg=4.27 nm, Dmax=18 nm) indicate very different shapes. The resulting models show a “tadpole”-like structure for the intact enzyme where the isotropic part coincides with the core protein and the flexible tail part should be identified with the C-terminal glycopeptide. Thus in this enzyme, functional differentiation is reflected in structural peculiarities.

[11] Small-angle x-ray scattering

Publisher Summary This chapter discusses small-angle scattering experiments with particles in solution—i.e., the particles are nonoriented. A large number of particles contribute to the scattering and the resulting spatial average leads to a loss in information. The information contained in the three-dimensional electron density distribution is thereby reduced to the one-dimensional distance distribution function. This function is proportional to the number of lines with length, which connect any volume element i with any volume element k of the same particle. The spatial orientation of these connection lines is of no account to the function. The connection lines are weighted by the product of the number of electrons situated in the volume elements i and k , respectively. The correlation between the function and the structure of the particle is also discussed in the chapter. The connection between the distance distribution function and the measured experimental scattering curve is also shown. It is observed that the each distance between two electrons of the particle, which is part of the function, leads to an angular-dependent scattering intensity. This physical process of scattering can be mathematically expressed by a Fourier transformation, which defines the way in which the information in “real space” (distance distribution function) is transformed into “reciprocal space” (scattering function). The chapter also discusses monochromatization and the camera type developed in Graz.

Investigation of cellobiohydrolase from trichoderma reesei by small angle X-ray scattering

SummaryTrichoderma reesei was grown on sulfite pulp and the major cellobiohydrolase of the culture filtrate was purified to homogeneity. The distance distribution function p(r) measured by the small angle X-ray scattering technique indicates that the enzyme molecule has a rather unusual tadpole like shape with an isotropic head and a long tail. The maximum length is 18 nm and the largest diameter is 4.4 nm.

Small-angle X-ray studies of Lumbricus terrestris haemoglobin

Abstract Small-angle X-ray scattering of Lumbricus terrestris haemoglobin was measured in dilute solutions in 0.1 M Tris HCl buffer, pH 7.0. The following molecular parameters were determined: radius of gyration 11.2 nm, volume 7700 nm3, maximum diameter 29 nm, molecular weight 3.95 × 106. The experimental scattering curve was compared with the scattering curves and distance distribution functions calculated for various models. The overall shape of the haemoglobin could be approximated by a hollow cylinder with the following dimensions: outer radius 13.5 nm, inner radius 5.4 nm, height 16.0 nm. The best fit was obtained with a model which consists of 12 large subunits arranged in two superimposed hexagonal rings with a number of smaller subunits between the large subunits and in the centre of the molecule.

Physico-chemical studies of fractionated bovine heparin: V. Some low-angle X-ray scattering data

Abstract The molecular weight and the radius of gyration of a fractionated sample of commercial bovine heparin were obtained using the method of low-angle X-ray scattering. The molecular weight of the heparin fraction was found to be 12,900 which is in excellent agreement with the values of 12,600 and 12,500 obtained for the same fraction from sedimentation equilibrium and viscosity measurements, respectively. A persistence length of 21.1 A and a radius of gyration of 35.9 A were calculated for the fraction. Under the experimental conditions studied (in water at room temperature) the heparin fraction may be described as a Gaussian coil molecule.

Small-angle X-ray scattering reveals differences between the quaternary structures of oxygenated and deoxygenated tarantula hemocyanin.

Small‐angle X‐ray scattering (SAXS) curves have been recorded for the oxygenated and deoxygenated states of the 4 × 6‐meric hemocyanin from the tarantula Eurypelma californicum. A comparison of the curves shows that the quaternary structures of the two states are different by three criteria, which all indicate that the hemocyanin is less compact in the oxygenated compared to the deoxygenated form: (a) The radius of gyration is 8.65 ± 0.05 nm for the deoxy‐ and 8.80 ± 0.05 nm for the oxy‐form. (b) The maximum particle dimension amounts to 25.0 ± 0.5 nm for the deoxy‐ and to 27.0 ± 0.5 nm for the oxyform. (c) A dip in the intramolecular distance distribution function p(r) is more pronounced and shifted to larger distances in the oxy‐form. The p(r) functions based on SAXS measurements were compared to p(r) functions deduced from published electron microscopical images of three different 4 × 6‐meric hemocyanins from closely related species. The p(r) functions of SAXS and electron microscopy were similar in one case, whereas in the other two cases the distance between the two 12‐meric half‐molecules had to be changed by 1–1.5 nm to obtain good agreement. The differences between the p(r) functions of oxygenated and deoxygenated 4 × 6‐meric tarantula hemocyanin are much larger than one would expect from a comparison of X‐ray structures of the oxygenated and deoxygenated states of a closely related 6‐meric hemocyanin. Thus, the conformational changes upon oxygenation occur at various levels of the quaternary structure, as postulated by hierarchical theories of allosteric interactions.

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.

Small-angle x-ray study of DNA-dependent RNA polymerase holoenzyme from Escherichia coli

RNA polymerase holoenzyme (Eo) of E. coli is a multisubunit enzyme composed of the subunits~~~~~u, The dissociable subunit (J is responsible for specific initiation [ 1,2]. After the synthesis of an RNA chain of a few nucleotides [3], CJ factor dissociates from the transcription complex (core RNA polymerase @‘@2), DNA and RNA), and can be used again as initiation factor. Here, the structure of holoenzyme (M, 487 000), the arrangement of u on core enzyme, is investigated by small-angle X-ray scattering, considering the results in [4,5] on the structure of core enzyme and CJ factor.

Small‐angle X‐ray study of DNA‐dependent RNA polymerase core enzyme and partial complex βα2 from Escherichia Coli

DNA-dependent RNA polymerase has to fulfill different complicated functions during transcription. For a detailed understanding of these functions, it is necessary to know the structure of this enzyme. The eubacterial RNA polymerase has the general composition formula /3’/302 (core enzyme) and /3’Polza (holoenzyme), respectively. The best characterized RNA polymerase is that of E. coli, which was used in our studies. Since protein crystals of RNA polymerase are not available, other methods were used for analysing the structure, such as electron microscopy [I], neutron [2,3] and X-ray small angle scattering [4]. Since, at that time the preparation and small angle scattering technique had not been developed as it is today, we describe here a new approach using X-ray scattering to study the structure of core enzyme (M, 395 000) and partial complex /301Z (M, /3 155 0OO;Mr cr2 73 000). For a detailed model of core enzyme it is necessary to know the structure of its subunits. The structure of isolated o2 has been investigated in [S]. Isolated subunits 0 and

Small-angle x-ray study of DNA-dependent RNA polymerase subunit α2 from Escherichia coli

are not suitable for X-ray scattering experiments, because of their tendency to aggregate. The study of the structure of the partial complex /3az, which can be homo-dispersly distributed in solution, will give information about the structure of /I. The aim of this paper is to verify the model of RNA polymerase core enzyme developed by neutron small angle scattering and to refine this model using information about the structure of isolated cr2 and