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Applications of the second virial coefficient: protein crystallization and solubility.

ABSTRACT: This article begins by highlighting some of the ground-based studies emanating from NASA's Microgravity Protein Crystal Growth (PCG) program. This is followed by a more detailed discussion of the history of and the progress made in one of the NASA-funded PCG investigations involving the use of measured second virial coefficients (B values) as a diagnostic indicator of solution conditions conducive to protein crystallization. A second application of measured B values involves the determination of solution conditions that improve or maximize the solubility of aqueous and membrane proteins. These two important applications have led to several technological improvements that simplify the experimental expertise required, enable the measurement of membrane proteins and improve the diagnostic capability and measurement throughput.

SUBMITTER: Wilson WW

PROVIDER: S-EPMC4014317 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Applications of the second virial coefficient: protein crystallization and solubility.

Wilson William W WW Delucas Lawrence J LJ

Acta crystallographica. Section F, Structural biology communications 20140430 Pt 5

This article begins by highlighting some of the ground-based studies emanating from NASA's Microgravity Protein Crystal Growth (PCG) program. This is followed by a more detailed discussion of the history of and the progress made in one of the NASA-funded PCG investigations involving the use of measured second virial coefficients (B values) as a diagnostic indicator of solution conditions conducive to protein crystallization. A second application of measured B values involves the determination of ...[more]

PMID: 24817708

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Project description:Antibody-based therapeutics are the fastest-growing drug class on the market, used to treat aggressive forms of cancer, chronic autoimmune conditions, and numerous other disease states. Although the specificity, affinity, and versatility of therapeutic antibodies can provide an advantage over traditional small-molecule drugs, their development and optimization can be much more challenging and time-consuming. This is, in part, because the ideal formulation buffer systems used for in vitro characterization inadequately reflect the crowded biological environments (serum, endosomal lumen, etc.) that these drugs experience once administered to a patient. Such environments can perturb the binding of antibodies to their antigens and receptors, as well as homo- and hetero-aggregation, thereby altering therapeutic effect and disposition in ways that are incompletely understood. Although excluded volume effects are classically thought to favor binding, weak interactions with co-solutes in crowded conditions can inhibit binding. The second virial coefficient (B2) parameter quantifies such weak interactions and can be determined by a variety of techniques in dilute solution, but analogous methods in complex biological fluids are not well established. Here, we demonstrate that fluorescence correlation spectroscopy is able to measure diffusive B2-values directly in undiluted serum. Apparent second virial coefficient (B2,app) measurements of antibodies in serum reveal that changes in the balance between attractive and repulsive interactions can dramatically impact global nonideality. Furthermore, our findings suggest that the approach of isolating specific components and completing independent cross-term virial coefficient measurements may not be an effective approach to characterizing nonideality in serum. The approach presented here could enrich our understanding of the effects of biological environments on proteins in general and advance the development of therapeutic antibodies and other protein-based therapeutics.

Project description:Interactions between proteins are often sufficiently weak that their study through the use of conventional structural techniques becomes problematic. Of the few techniques capable of providing experimental measures of weak protein-protein interactions, perhaps the most useful is the second virial coefficient, B(22), which quantifies a protein solution's deviations from ideal behavior. It has long been known that B(22) can in principle be computed, but only very recently has it been demonstrated that such calculations can be performed using protein models of true atomic detail (Biophys. J. 1998, 75:2469-2477). The work reported here extends these previous efforts in an attempt to develop a transferable energetic model capable of reproducing the experimental trends obtained for two different proteins over a range of pH and ionic strengths. We describe protein-protein interaction energies by a combination of three separate terms: (i) an electrostatic interaction term based on the use of effective charges, (ii) a term describing the electrostatic desolvation that occurs when charged groups are buried by an approaching protein partner, and (iii) a solvent-accessible surface area term that is used to describe contributions from van der Waals and hydrophobic interactions. The magnitude of the third term is governed by an adjustable, empirical parameter, gamma, that is altered to optimize agreement between calculated and experimental values of B(22). The model is applied separately to the proteins lysozyme and chymotrypsinogen, yielding optimal values of gamma that are almost identical. There are, however, clear difficulties in reproducing B(22) values at the extremes of pH. Explicit calculation of the protonation states of ionizable amino acids in the 200 most energetically favorable protein-protein structures suggest that these difficulties are due to a neglect of the protonation state changes that can accompany complexation. Proper reproduction of the pH dependence of B(22) will, therefore, almost certainly require that account be taken of these protonation state changes. Despite this problem, the fact that almost identical gamma values are obtained from two different proteins suggests that the basic energetic formulation used here, which can be evaluated very rapidly, might find use in dynamical simulations of weak protein-protein interactions at intermediate pH values.

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Applications of the second virial coefficient: protein crystallization and solubility.

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Applications of the second virial coefficient: protein crystallization and solubility.

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