Dieter Schubert

Size-Distribution Analysis of Proteins by Analytical Ultracentrifugation: Strategies and Application to Model Systems

Strategies for the deconvolution of diffusion in the determination of size-distributions from sedimentation velocity experiments were examined and developed. On the basis of four different model systems, we studied the differential apparent sedimentation coefficient distributions by the time-derivative method, g(s*), and by least-squares direct boundary modeling, ls-g*(s), the integral sedimentation coefficient distribution by the van Holde-Weischet method, G(s), and the previously introduced differential distribution of Lamm equation solutions, c(s). It is shown that the least-squares approach ls-g*(s) can be extrapolated to infinite time by considering area divisions analogous to boundary divisions in the van Holde-Weischet method, thus allowing the transformation of interference optical data into an integral sedimentation coefficient distribution G(s). However, despite the model-free approach of G(s), for the systems considered, the direct boundary modeling with a distribution of Lamm equation solutions c(s) exhibited the highest resolution and sensitivity. The c(s) approach requires an estimate for the size-dependent diffusion coefficients D(s), which is usually incorporated in the form of a weight-average frictional ratio of all species, or in the form of prior knowledge of the molar mass of the main species. We studied the influence of the weight-average frictional ratio on the quality of the fit, and found that it is well-determined by the data. As a direct boundary model, the calculated c(s) distribution can be combined with a nonlinear regression to optimize distribution parameters, such as the exact meniscus position, and the weight-average frictional ratio. Although c(s) is computationally the most complex, it has the potential for the highest resolution and sensitivity of the methods described.

Optimization of the preparation process for human serum albumin (HSA) nanoparticles

Nanoparticles prepared by desolvation and subsequent crosslinking of human serum albumin (HSA) represent promising carriers for drug delivery. Particle size is a crucial parameter, in particular for the in vivo behaviour of nanoparticles after intravenous injection. The objective of the present study is the development of a desolvation procedure for the preparation of HSA-based nanoparticles under the aspect of a controllable particle size between 100 and 300 nm in combination with a narrow size distribution. A pump-controlled preparation method was established which enabled particle preparation under defined conditions. Several factors of the preparation process, such as the rate of addition of the desolvating agent, the pH value and the ionic composition of the HSA solution, the protein concentration, and the conditions of particle purification were evaluated. The pH value of the HSA solution prior to the desolvation procedure was identified as the major factor determining particle size. Varying this parameter, (mean) particle diameters could be adjusted between 150 and 280 nm, higher pH values leading to smaller nanoparticles. Washing the particles by differential centrifugation led to significantly narrower size distributions. The reproducibility of the particle size and particle size distribution under the proposed preparation conditions was demonstrated by sedimentation velocity analysis in the analytical ultracentrifuge and the cellular uptake of those nanoparticles was studied by confocal microscope imaging and FACS analysis. The stability of the resulting nanoparticles was evaluated by pH and buffer titration experiments. Only pH values distinctly outside the isoelectric pH range of HSA and low salt concentrations were able to prevent nanoparticle agglomeration.

Comparison of scanning electron microscopy, dynamic light scattering and analytical ultracentrifugation for the sizing of poly(butyl cyanoacrylate) nanoparticles

Nanoparticles represent promising carriers for controlled drug delivery. This work focuses on the size and molecular mass characterization of polyalkylcyanoacrylate nanoparticles formed by anionic emulsion polymerization of butylcyanoacrylate in the presence of poloxamer 188 as a stabilizer. Three different methods were used to determine the size and size distribution of the particle populations: scanning electron microscopy (SEM), dynamic light scattering (DLS), and analytical ultracentrifugation (ANUC). SEM on freeze-dried and Au-shadowed samples showed a relatively narrow distribution of virtually spherical particles with a mean diameter of 167 nm. DLS yielded a monomodal distribution with hydrodynamic diameters around 199 nm (in the absence of additional stabilizer) or 184 nm (in the presence of 1% poloxamer 188). The size distribution determined by ANUC using sedimentation velocity analysis was somewhat more complex, the size of the most abundant particles being around 184 nm. Molar particle mass distributions centered around 2.3x10(9) g/mol. The advantages and disadvantages of the three sizing techniques are discussed.

Controlled Arrangement of Supramolecular Metal Coordination Arrays on Surfaces.

Metallo-supramolecular systems have been adsorbed in a controlled way onto graphite surfaces and visualized with molecular resolution for the first time. A parallel or orthogonal arrangement of the metal coordination arrays is evident depending on the specific ligands (see picture). Furthermore, simple nanomanipulations were performed by extracting single grids from the layer.

Projection structure and oligomeric properties of a bacterial core protein translocase

The major route for protein export or membrane integration in bacteria occurs via the Sec‐dependent transport apparatus. The core complex in the inner membrane, consisting of SecYEG, forms a protein‐conducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, purified and crystallized in two dimensions. A 9 Å projection structure was calculated using electron cryo‐microscopy. The structure exhibits P121 symmetry, having two asymmetric units inverted with respect to one another in the unit cell. The map shows elements of secondary structure that appear to be transmembrane helices. The crystallized form of SecYEG is too small to comprise the translocation channel and does not contain a large pore seen in other studies. In detergent solution, the SecYEG complex displays an equilibrium between monomeric and tetrameric forms. Our results therefore indicate that, unlike other known channels, the SecYEG complex can exist as both an assembled channel and an unassembled smaller unit, suggesting that transitions between the two states occur during a functional cycle.

The state of association of band 3 protein of the human erythrocyte membrane in solutions of nonionic detergents

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was solubilized and purified in aqueous solutions of two nonionic detergents: Ammonyx-LO (dimethyl laurylamine oxide) and C12E9 (nonaethylene glycol lauryl ether). The state of association of the purified protein was studied by analytical ultracentrifugation. Band 3 protein solubilized and studied in solutions of Ammonyx-LO was found to be in a monomer/dimer/tetramer association equilibrium. Band 3 protein freshly prepared in C12 E9 showed the same behaviour; however, during aging the protein was converted into stable noncovalent dimers. The conversion was retarded by the presence of beta-mercaptoethanol or by treatment of the samples with iodoacetamide; it seems to be due to oxidation of the protein by degradation products of the detergent. It is concluded that a monomer/dimer/tetramer association equilibrium is the native state of association of band 3 protein solubilized by nonionic detergents. Since nonionic detergents are assumed not to interfere with protein-protein interactions among membrane proteins, the results strongly support the claim that, in the erythrocyte membrane, band 3 is in a monomer/dimer/tetramer association equilibrium (Dorst, H.-J. and Schubert, D. (1979) Hoppe-Seylers Z. Physiol. Chem. 360, 1605-1618).

The nature of the stable noncovalent dimers of band 3 protein from erythrocyte membranes in solutions of Triton X-100

Stable noncovalent dimers of band 3 protein from human erythrocyte membranes, in which state the protein is thought to exist after solubilization by the nonionic detergent Triton X‐100, do not occur when purified batches of the detergent are used. Instead, the protein is in a monomer/dimer/tetramer association equilibrium. The stable dimers do appear, however, when the detergent has been ‘aged’. They thus seem to be artifacts.

Band 3-protein from human erythrocyte membranes strongly interacts with cholesterol

Band 3-protein [1], the main integral protein of the human erythrocyte membrane, is strongly involved in anion transport, the membranes most prominent function (for review, see ref. [2] ). Since, apparently, the biological function of an integral membrane protein is exerted by a combined action of the protein and neighbouring lipid and protein molecules (e.g., [3-5] ), efforts aimed at a better understanding of the mechanism of anion transport may benefit from studies on the interactions of the band 3-protein with other components of the erythrocyte membrane. Moreover, such studies are of obvious interest for the problem of erythrocyte membrane structure. Several investigations have already been performed on the protein-protein interactions of the band 3-protein [1,6,7]. No information is available, however, on the interactions of this protein with lipids. Recently, we have developed a new method for the isolation of the band 3-protein which avoids the use of detergents [8]. Using this method of protein preparation, we were now able to study the interactions of the protein with lipid monolayers at the air-water interface. These experiments, which are described in this paper, have revealed a strong interaction of the band 3-protein with cholesterol.

Band 3 protein—cholesterol interactions in erythrocyte membranes: Possible role in anion transport and dependency on membrane phospholipid

Band 3 protein of the human erythrocyte membrane, the anion transport protein, possesses a high affinity steroid binding site. In mixed phospholipid—cholesterol monolayers, the state of occupancy of this site is positively correlated with their cholesterol and sphingomyelin content and negatively with their glycerophospholipid content. We suggest that, in the erythrocyte membrane, the binding site is an inhibitory site of anion transport and that the modulation of its state of occupancy by the membrane lipid is responsible for the negative correlation of anion transport with the membranes content of cholesterol and sphingomyelin and the positive correlation with the phosphatidylcholine content

Band 3‐hemoglobin associations The band 3 tetramer is the oxyhemoglobin binding site

The associations between the band 3 protein of the human erythrocyte membrane and oxyhemoglobin, in solutions of a nonionic detergent, were studied by sedimentation equilibrium experiments in the analytical ultracentrifuge. The following results were obtained: (i) hemoglobin is bound virtually exclusively to the band 3 tetramer, but not to the monomer or dimer; (ii) the band 3 tetramer can bind up to four hemoglobin tetramers; (iii) unlike the unstable dimers of unmodified band 3, stable dimers crosslinked via S S‐bridges also represent hemoglobin binding sites.