Helmut Durchschlag

Computational and Experimental Evidence for the Evolution of a (βα) 8 -Barrel Protein from an Ancestral Quarter-Barrel Stabilised by Disulfide Bonds

The evolution of the prototypical (beta alpha)(8)-barrel protein imidazole glycerol phosphate synthase (HisF) was studied by complementary computational and experimental approaches. The 4-fold symmetry of HisF suggested that its constituting (beta alpha)(2) quarter-barrels have a common evolutionary origin. This conclusion was supported by the computational reconstruction of the HisF sequence of the last common ancestor, which showed that its quarter-barrels were more similar to each other than are those of extant HisF proteins. A comprehensive sequence analysis identified HisF-N1 [corresponding to (beta alpha)(1-2)] as the slowest evolving quarter-barrel. This finding indicated that it is the closest relative of the common (beta alpha)(2) predecessor, which must have been a stable and presumably tetrameric protein. In accordance with this prediction, a recombinantly produced HisF-N1 protein was properly folded and formed a tetramer being stabilised by disulfide bonds. The introduction of a disulfide bond in HisF-C1 [corresponding to (beta alpha)(5-6)] also resulted in the formation of a stable tetramer. The fusion of two identical HisF-N1 quarter-barrels yielded the stable dimeric half-barrel HisF-N1N1. Our findings suggest a two-step evolutionary pathway in which a HisF-N1-like predecessor was duplicated and fused twice to yield HisF. Most likely, the (beta alpha)(2) quarter-barrel and (beta alpha)(4) half-barrel intermediates on this pathway were stabilised by disulfide bonds that became dispensable upon consolidation of the (beta alpha)(8)-barrel.

Calculation of Partial Specific Volumes and Other Volumetric Properties of Small Molecules and Polymers

Volumetric properties of macromolecules and low-molecular compounds are necessary auxiliary means for the determination of molar masses from solution-scattering and hydrodynamic techniques. In many cases experimental determinations of partial volumes can or have to be replaced by calculative procedures. A universal approach for the calculation of both low-molecular organic compounds and polymers of different chemical composition and structure in aqueous solution is described. It is based on volume increments for the constituent atoms, ions and/or groups and allows corrections for covolume, ring formation, micellization, ionization etc. Application of this approach is of particular interest in connection with: (i) nonionic and ionic organic solutes; (ii) inorganic electrolytes; (iii) monomeric and micellar detergents and lipids; (iv) carbohydrates and polysaccharides; (v) nucleobases, nucleosides, nucleotides, polynucleotides, nucleic acids; (vi) amino acids, amino-acid residues, peptides, polyaminoacids, nonconjugated and conjugated proteins; (vii) synthetic polymers. The results of the volume predictions show a high degree of reliability, if compared to experimental data. Special approaches dealing with simple and conjugated proteins and protein-ligand complexes in two- and multicomponent solutions allow the prediction of both isomolal and isopotential volumes under a variety of native and denaturing conditions, including the presence of high amounts of additives.

Partial specific volume changes of proteins densimetric studies

Abstract The partial specific volume of proteins has been investigated as a function of protein concentration, temperature, pH, and a number of denaturing and non-denaturing solvent components. Protein concentration (5–100 mg·ml −1 ) is found to have no effect; increasing temperature (4–40°C), as well as addition of electrolytes, sugars, and polyols, leads to an approximately linear increase; upon ligand binding, as well as denaturation by heat, extremes of pH, guanidine. HCl, urea, or sodium dodecyl sulfate, a predominantly non-linear decrease is observed. The study demonstrates the importance of measuring rather than calculating the specific volume of proteins. This holds especially in multicomponent systems, where solvent conditions may cause drastic effects.

Comparative investigations of biopolymer hydration by physicochemical and modeling techniques

The comparative investigation of biopolymer hydration by physicochemical techniques, particularly by small-angle X-ray scattering, has shown that the values obtained differ over a wide range, depending on the nature of the polymer and the environmental conditions. In the case of simple proteins, a large number of available data allow the derivation of a realistic average value for the hydration (0.35 g of water per gram of protein). As long as the average properties of proteins are considered, the use of such a default value is sufficient. Modeling approaches may be used advantageously, in order to differentiate between different assumptions and hydration contributions, and to correctly predict hydrodynamic properties of biopolymers on the basis of their three-dimensional structure. Problems of major concern are the positioning and the properties of the water molecules on the biopolymer surface. In this context, different approaches for calculating the molecular volume and surface of biopolymers have been applied, in addition to the development of appropriate hydration algorithms.

Comparative investigations of the effects of X- and UV-irradiation on lysozyme in the absence or presence of additives

Abstract The radiation damage of lysozyme has been investigated in aqueous solution, after preceding X- and UV-irradiation in the absence or presence of various additives. Activity measurements and UV absorption and fluorescence spectroscopy turned out to be powerful screening techniques for disclosing the damages (inactivation, aggregation, loss of aromatics) and modifications (protection) provided by many additives. Advanced computer modeling approaches were used to visualize the protein atoms and the water molecules bound preferentially on the protein surface. Correlations between the position of radiosensitive amino acid residues and occurring radiation damages are discussed.

Detection of small conformational changes of proteins by small‐angle scattering

In the past the technique of small-angle scattering has been a powerful tool for studying conformational changes of proteins which occur, for example, upon binding with ligands. Results obtained by different authors from X-ray and neutron experiments on a variety of proteins and under various conditions have been compiled. This offers the possibility of comparing the extent of changes in the molecular parameters investigated (e.g. change of the radius of gyration). Problems encountered with the detection of small changes are discussed. As an example, conformational changes of the enzyme citrate synthase upon substrate binding (oxaloacetate) are presented. X-ray crystallography had already found distinct changes between open and closed forms of the enzyme. Small-angle X-ray scattering studies registered slight changes of some parameters in solution. These changes could be paralleled with the results of other solution techniques (UV absorption, fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation). The results found for citrate synthase are also compared with previous findings for malate synthase, an enzyme of similar enzymatic function. Above all, this study shows that care has to be taken when studying small conformational changes. It is absolutely necessary to use different methods and conditions and to study the problem from different points of view to avoid pitfalls.

Small-angle X-ray studies on malate synthase from Baker's yeast

Abstract Malate synthase was investigated in solution by the small-angle X-ray scattering technique. The substrate-free enzyme was shown to have a molecular weight of 186000, a radius of gyration of 3.96 nm, a maximum particle diameter of 11.2 nm, a volume of 343 nm3, a radius of gyration of the thickness of 1.04 nm, and an axial ratio of 1:0.33. The enzyme molecule undergoes small changes in overall structure upon binding substrates. Investigation of the enzyme under prolonged exposure to X-rays led to an aggregation of the enzyme and allowed statements concerning the way of aggregation and factors influencing aggregation.

Modeling of protein solution structures

Reconstructions of low-resolution three-dimensional solution structures of proteins from one-dimensional small-angle X-ray scattering (SAXS) data can be achieved by trial-and-error or ab initio approaches. It is demonstrated that all tested advanced approaches (GA, DAMMIN, SAXS3D) reproduce the shape of three selected test proteins (malate synthase, cellobiose dehydrogenase, extracellular hemoglobin). The agreement between SAXS profiles and functions derived from beaded model structures and experimental data, however, varies with respect to the angular range covered; the same holds true for the accuracy of the molecular parameters calculated from the respective patterns. The uniqueness of the structures obtained is crucial and may be increased by combined use of different approaches. The obtained models can be used for predicting both structural and hydrodynamic data with a high degree of probability.

Comparative studies of structural properties and conformational changes of proteins by analytical ultracentrifugation and other techniques

Analytical ultracentrifugation is a powerful tool for investigating the size of proteins in solution, especially by measuring sedimentation and diffusion coefficients and molar masses. Several further molecular parameters such as frictional ratios, axial ratios of hydrodynamic models, and Stokes radii allow a rough estimate of the protein overall structure. Sedimentation analysis may also be applied efficaciously for monitoring conformational changes of proteins occurring upon ligand binding or denaturation. For the determination of very small changes in shape, however, great care and a series of precautions are required. We investigated the enzymes citrate synthase and malate synthase in the absence and in the presence of ligands, in order to study the structural properties of the proteins and their ligand complexes. We also compared the results of the ultracentrifugal analysis with the results of other solution techniques such as UV absorption, fluorescence spectroscopy, circular dichroism, and small-angle x-ray scattering on the one hand, and the crystallographic 3D structure of citrate synthase on the other. The spectroscopic methods may be used as efficient and rapid tools for screening the occurrence of conformational changes caused by alterations of chromophores and fluorophores. The structural information provided by small-angle scattering (e.g., radii of gyration, maximum particle diameters, vclumes and surface areas) can be used to establish quantitative correlations between solution scattering and hydrodynamic data. In this context, however, knowledge or qualified assumptions of partial specific volumes and hydration are additionally required. Good agreement was reached between small-angle scattering and ultracentrifugal data, and also with crystallographic data if protein hydration was considered properly. The given approaches may be used to predict hydrodynamic properties if x-ray data are available, and for many verifications of other structural data, e.g., Stokes radii, diffusion coefficients, axial and frictional ratios determined by independent methods.

Molecular structure of malate synthase and structural changes upon ligand binding to the enzyme

Abstract Malate synthase has a molecular weight of about 170 000 as shown by ultracentrifugation, sucrose gradient centrifugation, and thin layer gel-chromatography. High dilution, extremes of pH, succinylation, and treatment with sodium dodecylsulfate suggest the enzyme to be a tetramer. The CD spectrum is typical for a globular protein with moderate helical content (∼30 %), and shows anomalous Cotton effects at 250–290 nm. Binding of substrates (acetyl-CoA, glyoxylate) or the substrate analog pyruvate causes slight conformational changes which are reflected in alterations of the CD bands in the range of aromatic absorption; binding of Mg2+ causes no structural effects, suggesting the metal ion to be involved in enzymatic catalysis rather than structural alterations.