Martin Zulauf

Light scattering of proteins as a criterion for crystallization

Abstract Light scattering is particularly sensitive for the detection of aggregates as the scattered intensity is proportional to the square of the molecular weight of the scattering molecules. We have found that proteins showing a tendency to form aggregates in dilute solution (and in the absence of precipitating agents) do not crystallize in the majority of cases. Thus detection of aggregates seems to indicate that crystallization will not be successful. Fifteen proteins have been studied using this method to determined the correlation between aggregation and crystallization.

The micelle to vesicle transition of lipids and detergents in the presence of a membrane protein: towards a rationale for 2D crystallization

The assembly of two‐dimensional membrane protein crystals in the presence of lipids was analyzed with quasielastic light scattering and electron microscopy. Mixtures of detergent‐solubilized lipids and/or proteins were submitted to slow or rapid dilution while measuring the hydrodynamic radii of the aggregates. Lipids alone exhibited λ‐shaped dilution curves with intermediate rod‐shaped particles that converted into small vesicles. Depending on the protein‐protein and protein‐lipid interactions, detergent‐solubilized protein‐lipid mixtures showed a sharp transition from micelles to large, densely packed proteoliposomes. Electron microscopy revealed that formation of crystals occurred shortly after this phase transition.

Assembly of 2-D membrane protein crystals: dynamics, crystal order, and fidelity of structure analysis by electron microscopy.

Membrane protein reconstitution into two-dimensional (2-D) ordered arrays is described. The assembly of 2-D crystals may be modeled as a two-step process: the membrane protein is first integrated in the lipid bilayer and then crystallized by removal of excess detergent or lipid and/or by precipitating agents. Lipid-detergent, protein-detergent, and lipid-protein interactions are critical during the first step, while lipid-protein and protein-protein interactions dominate events in the second step. The evidence supporting this model results from quasielastic light scattering analyses and electron microscopy of different lipid-detergent systems and reconstitution experiments with Escherichia coli porin OmpF, Phormidium laminosum photosystem I reaction centers, and integral membrane proteins of mammalian lens fiber cells.

Tocopherol transport and absorption

Vitamin E is the generic term for a group of lipid-soluble tocol and tocotrienol derivatives possessing varying degrees of vitamin activity. The most active of these compounds is a-tocopherol, which accounts for approximately 90% of the vitamin E found in tissues. The present paper will, therefore, principally consider the absorption and transport of a-tocopherol from ingestion to uptake by tissues, including a-tocopheryl acetate, which is the most widely used form for vitamin E supplementation. Tocopherols are water insoluble, non-swelling amphiphiles. Thus, movement through the aqueous phase at appropriate concentrations depends on the abundancy of suitable vehicles enabling efficient vitamin E absorption and transport.

Molecular weight of adenovirus serotype 2 capsomers. A new characterization.

Abstract We have determined the molecular weight of some of the adenovirus serotype 2 structural proteins: penton, penton base and fibre. Physical techniques, namely neutron scattering and hydrodynamical measurements, indicate that the penton base is a trimer. This is confirmed by analysis of the virion composition based on quantitative gel scanning. This finding implies either that other proteins (e.g. protein IIIa) are essential in the architecture of the fivefold vertex of the virion, or that the usual assumption that icosahedral symmetry involves identical interactions related to the symmetry of the virion does not hold.

Interferon gamma receptor extracellular domain expressed as IgG fusion protein in Chinese hamster ovary cells. Purification, biochemical characterization, and stoichiometry of binding.

Agents that antagonize the functions of interferon γ (IFNγ) are potential pharmaceuticals against several immunological and inflammatory disorders. IFNγ receptor-immunoglobulin G fusion proteins (IFNγR-IgG) function as antagonists of endogenous IFNγ and have longer half-lives in vivo in comparison with soluble IFNγ receptors (sIFNγR), consisting of the extracellular region of the native sequence. A fusion protein comprising the extracellular domain of the human IFNγ receptor and the hinge, CH2 and CH3 domains of the human IgG3 constant region, was expressed in Chinese hamster ovary cells. The IFNγR-IgG3 fusion protein was secreted into the culture medium as a 175-kDa glycoprotein and was purified over Protein G-Sepharose, DEAE-Sepharose, and size exclusion chromatography. IFNγR-IgG3 bound IFNγ in solid phase assays and ligand blots, competed for the binding of radiolabeled IFNγ to the cell surface receptor of Raji cells, and inhibited the IFNγ-mediated antiviral activity with an efficiency at least one order of magnitude higher than that of the soluble receptor produced in the same expression system. Two IFNγR-IgG3 fusion proteins bound two IFNγ dimers forming a complex of approximately 380 kDa. In immunodiffusion assays, the IFNγR-IgG3 fusion protein did not precipitate IFNγ. Dissociation of bound IFNγ from IFNγR-IgG3 was 2-fold slower than from the sIFNγR produced in insect cells.

Expression, purification and crystallization of fully active, glycosylated human interleukin-5

Recombinant human interleukin‐5 (hIL‐5) has been expressed at high levels and produced in large quantities in baculovirus infected Sf9 insect cells. The glycosylated protein was purified using immuno‐affinity chromatography and gel filtration. Purified hIL‐5 has been crystallized using standard vapour diffusion techniques with PEG as a coprecipitant. The crystals belong to the C2 space group and diffract to 2 Å.

Density determination by analytical ultracentrifugation in a rapid dynamical gradient: application to lipid and detergent aggregates containing proteins

A rapidly developing dynamical gradient can be formed in the analytical centrifuge when a buffer solution prepared in D2O is underlayered under the same buffer solution prepared in H2O in a specially designed double sector cell. In a short time the boundary layer spreads to form the gradient. Heavy particles (S greater than or equal to 10S) will band in the gradient corresponding to their density, which can be determined accurately. To this end, the buffer may contain density adjusting additives such as sucrose. We present results with this technique for lipid vesicles, for a Ca-antagonist bound to vesicles, as well as for a lipoprotein and reconstituted regular membrane protein-lipid arrays.

Light Scattering Techniques

The aim of light scattering experiments with solutions of macromolecules is to determine their size and shape. However, as a matter of fact, interactions among the macromolecules also affect light scattering, and the retrieval of information on size and shape may be complicated. This comes as follows. To analyze light scattering data, one has to solve a theoretical problem first: given a set of particles of known size, shape and optical properties, and distributed in the solvent in a defined way, find the field at all space points when they are illuminated by a monochromatic wave of arbitrary polarization. This problem entails the solution of Maxwell’s equations with given boundary conditions. In the so-called Rayleigh-Debye-Gans approximation, which holds when the refractive index difference between particles and solvent is small compared to the solvent index and when the phase difference between a wave propagating inside the particle and one propagating outside is small in terms of the wavelength of the light, the problem reduces to the evaluation of interferences between scattering amplitudes emanating from all the volume elements in space. For spherical particles the result is easy to write down.