Bulletin of the American Physical Society
APS March Meeting 2010
Volume 55, Number 2
Monday–Friday, March 15–19, 2010; Portland, Oregon
Session Q5: A Critical Challenge for the Biotech Industry: The Measurement of Protein Associations |
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Sponsoring Units: FIAP Chair: Philip Wyatt, Wyatt Technology Corporation Room: Portland Ballroom 256 |
Wednesday, March 17, 2010 11:15AM - 11:51AM |
Q5.00001: Plasmon Spectroscopy Applied to Biomolecular Interactions in Membranes Invited Speaker: Plasmon-waveguide resonance (PWR) is an optical spectroscopy method that can provide information about materials immobilized on the surface of a plasmon resonator consisting of a right angle prism coated with thin layers of a metal (approx. 50 nm; usually silver) and a dielectric (approx. 500 nm; usually silica). The technique has been developed in our laboratory and is an extension of the more commonly used surface plasmon resonance (SPR) method, having higher sensitivity (20-50 fold) and resolution (10-20 fold). The dielectric layer allows plasmon excitation by light whose electric vector is polarized \underline {both} perpendicular and parallel to the sensor surface, in contrast to SPR that can only utilize perpendicular polarized excitation. This allows \underline {both} mass density and mass distribution to be characterized in uniaxially oriented deposited materials, such as biomembranes. We have utilized this technique to investigate binding interactions between membrane-incorporated protein receptors and their ligands (both proteins and small molecules), using both purified receptors inserted into lipid bilayers and membranes derived from cells expressing these receptors. Such studies have provided many new insights into biological signaling events. Inasmuch as many of these receptors are targets for approximately 50 percent of ethical drugs, PWR can be a useful methodology for drug discovery in the pharmaceutical industry. Examples of these experiments will be presented. [Preview Abstract] |
Wednesday, March 17, 2010 11:51AM - 12:27PM |
Q5.00002: Protein associations and analytical ultracentrifugation Invited Speaker: Analytical ultracentrifugation (AUC) is a first principle method for characterizing the thermodynamics of macromolecules in solution. Since AUC directly assesses mass, it is particularly useful for characterizing both reversible and irreversible binding interactions between macromolecules. The principle measurement in AUC is the concentration as a function of radial position, which may be provided by either absorbance, interference or fluorescence detection. Each of these three different detectors may be used to characterize protein associations using either sedimentation equilibrium or sedimentation velocity analysis. Examples will be shown for characterizing irreversible (aggregate) formation, high-accuracy reversible association analysis, and the detection of protein interactions in complex and concentrated fluids (e.g. serum, cell cytosol). [Preview Abstract] |
Wednesday, March 17, 2010 12:27PM - 1:03PM |
Q5.00003: Quantitative Characterization of Protein Associations in Highly Concentrated Solution Invited Speaker: With few exceptions, one cannot reliably predict the behavior of a protein at high concentration on the basis of knowledge obtained from experiments carried out at low concentration. Detection and quantitative characterization of protein-protein interactions in the high concentration regime ($>$ 50 g/L) therefore presents both experimental and theoretical challenges to the investigator. Two experimental methods devised in our laboratory specifically for this purpose are described. (1) \textit{Non-ideal tracer sedimentation equilibrium.} Instrumentation and theory for measuring and interpreting the equilibrium gradient of a labeled dilute tracer protein in a solution containing an arbitrary concentration of one or more unlabeled macromolecules are outlined. The composition dependence of the equilibrium gradient of several proteins, including ribonuclease at concentrations up to 200 g/L and immunoglobulin G at concentrations up to 125 g/L, will be presented and interpreted in the context of models taking into account both equilibrium self-association, and nonspecific repulsive steric or electrostatic repulsion. (2) \textit{Non-ideal light scattering. }Recently developed instrumentation and theory for rapid measurement and interpretation of the light scattering of a protein solution over a broad range of concentration are outlined. The concentration-dependent light scattering of chymotrypsin A at three different pH values at concentrations up to 60 g/L, and the concentration-dependent light scattering of two monoclonal antibodies at concentrations up to over 200 g/L in solutions of varying ionic strength, are quantitatively accounted for by models that take into account both nonideal repulsion between protein molecules and specific modes of equilibrium self-association. [Preview Abstract] |
Wednesday, March 17, 2010 1:03PM - 1:39PM |
Q5.00004: Composition-Gradient Static Light Scattering and the Quantification of Biomolecular Interactions in Therapeutic Proteins Invited Speaker: Macromolecular interactions of interest to the pharmaceutical industry cover a variety of phenomena: binding of proteins to form well-defined complexes; reversible and irreversible oligomerization; and non-specific intermolecular interactions. The analysis and manipulation of these phenomena are crucial to the successful development, manufacture, storage and delivery of biological drugs such as antibodies. Light scattering (LS) has proven to be one of the most versatile free-solution and label-free methods for studying proteins and their interactions. Previously limited primarily to assessing molar mass, size and oligomerization state, the recent emergence of automated Composition-Gradient Static Light Scattering (Attri, A.; Minton, A.P. \textit{Anal. Biochem. }\textbf{2005}; $346$(1), 132--8), or CG-SLS, extends the range of biotech LS applications to equilibrium binding affinity and stoichiometry of bound complexes, kinetics of association and dissociation, and non-specific interactions (attractive and repulsive). In this talk I present progress in CG-SLS for biophysical characterization of pharmaceutical protein-protein interactions. In the drug development phase, CG-SLS studies of antibody-antigen complexes compliments other biomolecular interaction techniques commonly found in the biotech world such as surface plasmon resonance (SPR). In the formulation development stage, long-term stability of drug product is sought. Protein degradation modes include irreversible aggregation, which may lead to adverse physiological effects, and reversible self-association, which affects solution viscosity and hence injectable drug delivery. CG-SLS addresses both of these, the former via determination of virial coefficients, which describe the overall non-specific attraction or repulsion between molecules and may be used to optimize the formulation buffer to minimize aggregation, and the latter by binding affinity and stoichiometry of the associated complexes. [Preview Abstract] |
Wednesday, March 17, 2010 1:39PM - 2:15PM |
Q5.00005: Inhibition of Protein-Protein Interactions and Signaling by Small Molecules Invited Speaker: Protein-protein interactions are at the core of cell signaling pathways as well as many bacterial and viral infection processes. As such, they define critical targets for drug development against diseases such as cancer, arthritis, obesity, AIDS and many others. Until now, the clinical inhibition of protein-protein interactions and signaling has been accomplished with the use of antibodies or soluble versions of receptor molecules. Small molecule replacements of these therapeutic agents have been extremely difficult to develop; either the necessary potency has been hard to achieve or the expected biological effect has not been obtained. In this presentation, we show that a rigorous thermodynamic approach that combines differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) provides a unique platform for the identification and optimization of small molecular weight inhibitors of protein-protein interactions. Recent advances in the development of cell entry inhibitors of HIV-1 using this approach will be discussed. [Preview Abstract] |
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