W. William Wilson

Predicting protein crystallization from a dilute solution property

A dilute solution parameter obtained from static light-scattering measurements is proposed as a predictor for protein crystallization experiments. The osmotic second virial coefficients, B(22), have been measured for a variety of proteins in solvents that are known to promote crystallization and the values for B(22) were found to lie within a fairly narrow range which we refer to as a crystallization slot. Solution conditions which were known not to favor crystallization of the proteins resulted in B(22) values well outside the crystallization slot.

Status of methods for assessing bacterial cell surface charge properties based on zeta potential measurements

Surface interfacial physiology is particularly important to unicellular organisms with regard to maintenance of optimal cell function. Bacterial cell surfaces possess net negative electrostatic charge by virtue of ionized phosphoryl and carboxylate substituents on outer cell envelope macromolecules which are exposed to the extracellular environment. The degree of peripheral electronegativity influences overall cell surface polarity and can be assessed on the basis of zeta potential which is most often determined by estimating the electrophoretic mobility of cells in an electric field. The purpose of this review is to provide bacteriologists with assistance as they seek to better understand available instrumentation and fundamental principles concerning the estimation of zeta potential as it relates to bacterial surface physiology.

Correlation of Second Virial Coefficients and Solubilities Useful in Protein Crystal Growth

Abstract The osmotic second virial coefficient, B , is a dilute solution parameter, so one may wonder what relationships exist among B and the many facets of protein crystal growth (PCG). In particular, knowledge of how a proteins solubility ( S ) depends on solution variables such as temperature, pH and ionic strength is very useful for finding optimum conditions for the PCG experiment. In this work, a correlation between the second virial coefficient and protein solubility is shown for ovalbumin and lysozyme solutions. The data presented suggest significant correlation between B and S as a function of crystallizing salt type and concentration, pH and temperature. The data are presented both in graphical and tabular form so that it maybe useful to researchers desiring to develop and test detailed models for protein interactions.

Light scattering as a diagnostic for protein crystal growth—A practical approach

Static and dynamic light scattering are discussed as particularly useful tools for studying various aspects of protein crystal growth. Specific applications for prenucleation assays as well as for monitoring postnucleation growth processes are presented. Protein-protein interactions determined by light scattering, which serve as a predictor for favorable crystallization conditions as well as for protein solubility behavior, are detailed. Several precautions regarding the practical aspects of light scattering and interpretation of data are also discussed.

Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution.

There has been an increasing awareness that proteins, like other biopolymers, are large enough to exhibit colloidal behavior in aqueous solution. Net attractive or repulsive forces have been found to govern important physical properties, such as solubility and aggregation. The extent of intermolecular interactions, usually expressed in terms of the osmotic second virial coefficient, B, is most often measured using static light scattering. More recently, self-interaction chromatography (SIC) has emerged as a method for rapid determination of B in actual formulations, as it uses much less protein and has higher throughput. This review will summarize the relationship of B to crystallization, solubility, and aggregation of proteins in aqueous solution. Moreover, the capability of SIC to obtain B values in a rapid and reproducible fashion will be described in detail. Finally, the use of miniaturized devices to measure B is presented.

Contribution of Choline-Binding Proteins to Cell Surface Properties of Streptococcus pneumoniae

ABSTRACT Nonspecific interactions related to physicochemical properties of bacterial cell surfaces, such as hydrophobicity and electrostatic charge, are known to have important roles in bacterium-host cell encounters. Streptococcus pneumoniae (pneumococcus) expresses multiple, surface-exposed, choline-binding proteins (CBPs) which have been associated with adhesion and virulence. The purpose of this study was to determine the contribution of CBPs to the surface characteristics of pneumococci and, consequently, to learn how CBPs may affect nonspecific interactions with host cells. Pneumococcal strains lacking CBPs were derived by adapting bacteria to a defined medium that substituted ethanolamine for choline. Such strains do not anchor CBPs to their surface. Cell surface hydrophobicity was tested for the wild-type and adapted strains by using a biphasic hydrocarbon adherence assay, and electrostatic charge was determined by zeta potential measurement. Adherence of pneumococci to human-derived cells was assessed by fluorescence-activated cell sorter analysis. Strains lacking both capsule and CBPs were significantly more hydrophobic than nonencapsulated strains with a normal complement of CBPs. The effect of CBPs on hydrophobicity was attenuated in the presence of capsule. Removal of CBPs conferred a greater electronegative net surface charge than that which cells with CBPs possessed, regardless of the presence of capsule. Strains that lack CBPs were poorly adherent to human monocyte-like cells when compared with wild-type bacteria with a full complement of CBPs. These results suggest that CBPs contribute significantly to the hydrophobic and electrostatic surface characteristics of pneumococci and may facilitate adherence to host cells partially through nonspecific, physicochemical interactions.

Correlation between the osmotic second virial coefficient and solubility for equine serum albumin and ovalbumin

The Haas - Drenth - Wilson (HDW) (Haas et al., 1999) theoretical model was used to correlate osmotic second virial coefficient (B) values with solubility (S) values for equine serum albumin (ESA) and ovalbumin for corresponding solution conditions. The best fit from the theoretical model was compared to experimental S versus B data. B values were experimentally measured using static light scattering. Solubilities of ESA were estimated using a sitting drop method. When the experimental data for S versus B were plotted, an excellent fit for ESA was obtained according to the HDW model. The results showed that the coordination number (z) in the crystal lattice was 6, and the adjustable parameter (A) was 0.072. For ovalbumin, previously reported solubility data in aqueous ammonium sulfate solutions were utilized. The solubility data for ovalbumin were correlated with the measured B values obtained in our laboratory. The resulting best fit from the HDW model showed that z = 6 and A = 0.084.

Temperature-independent solubility and interactions between apoferritin monomers and dimers in solution

Abstract We used chromatographic, static and dynamic light scattering techniques, and atomic force microscopy (AFM) to study the structure of the protein species and the protein–protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of NaAc buffer and CdSO 4 . The sizes and shapes of the monomers and dimers, separated by size-exclusion chromatography, were determined by dynamic light scattering and AFM. While the monomer is an apparent sphere with a diameter corresponding to previous X-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was used to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The addition of even small amounts of Cd 2+ causes attraction between the monomer molecules. Furthermore, we found that the second virial coefficient and the protein solubility do not noticeably depend on temperature in the range from 0°C to 40°C. This suggests that the enthalpy for crystallization of apoferritin is close to zero, and the gain of entropy is essentially constant in this temperature range. We also found that in solutions of the apoferritin dimer, the molecules attract even in the presence of acetate buffer only, indicating a change in the surface of the apoferritin molecule. In view of the repulsion between the monomers at the same conditions, this suggests that the dimers and higher oligomers form only after partial unfolding of some of the apoferritin subunits. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization in the protein solution.

A fiber optic probe for monitoring protein aggregation, nucleation and crystallization

Protein crystals are often experimentally grown in hanging drops in microgravity experiments on-board the Space Shuttle orbiter. The technique of dynamic light scattering (DLS) can be used to monitor crystal growth processes in hanging droplets (∼ 30 μL) in microgravity experiments, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. In this paper we demonstrate that such experiments are now feasible. We apply a newly developed fiber optic probe to various earth and space (microgravity) protein crystallization system configurations to test its capabilities. These include conventional batch (cuvette or capillary) systems, a hanging drop method in a six-pack hanging drop vapor diffusion apparatus (HDVDA), a modified HDVDA for temperature-induced nucleation and aggregation studies, and a newly envisioned dynamically controlled vapor diffusion system (DCVDS) configuration. Our compact system exploits the principles of DLS and offers a fast (within a few seconds) means of quantitatively and non-invasively monitoring the various growth stages of protein crystallization. In addition to DLS capability, the probe can also be used for performing single-angle static light scattering measurements. It utilizes extremely low levels of laser power (a few μW) and essentially eliminates the usual problems associated with optical alignment and vibration isolation. The compact probe is also equipped with a miniaturized microscope for visualization of macroscopic protein crystals. This new optical diagnostic system makes possible the exploration of new ways to grow good quality crystals suitable for X-ray crystallographic analysis and may contribute to a concrete scientific basis for understanding the process of crystallization.

Measuring Protein Interactions by Microchip Self-Interaction Chromatography

The self‐interaction of proteins is of paramount importance in aggregation and crystallization phenomena. Solution conditions leading to a change in the state of aggregation of a protein, whether amorphous or crystalline, have mainly been discovered by the use of trial and error screening of large numbers of solutions. Self‐interaction chromatography has the potential to provide a quantitative method for determination of protein self‐interactions amenable to high‐throughput screening. This paper describes the construction and characterization of a microchip separation system for low‐pressure self‐interaction chromatography using lysozyme as a model protein. The retention time was analyzed as a function of mobile‐phase composition, amount of protein injected, flow rate, and stationary‐phase modification. The capacity factors (k′) as a function of crystallizing agent concentration are compared with previously published values for the osmotic second virial coefficient (B22) obtained by static light scattering, showing the ability of the chip to accurately determine protein‐protein interactions. A 500‐fold reduction in protein consumption and the possibility of using conventional instrumentation and automation are some of the advantages over currently used methodologies for evaluating protein‐protein interactions.