Using Lamm-Equation modeling of sedimentation velocity data to determine the kinetic and thermodynamic properties of macromolecular interactions

Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-8816, USA.
Methods (Impact Factor: 3.65). 12/2010; 54(1):4-15. DOI: 10.1016/j.ymeth.2010.12.029
Source: PubMed


The interaction of macromolecules with themselves and with other macromolecules is fundamental to the functioning of living systems. Recent advances in the analysis of sedimentation velocity (SV) data obtained by analytical ultracentrifugation allow the experimenter to determine important features of such interactions, including the equilibrium association constant and information about the kinetic off-rate of the interaction. The determination of these parameters is made possible by the ability of modern software to fit numerical solutions of the Lamm Equation with kinetic considerations directly to SV data. Herein, the SV analytical advances implemented in the software package SEDPHAT are summarized. Detailed analyses of SV data using these strategies are presented. Finally, a few highlights of recent literature reports that feature this type of SV data analysis are surveyed.

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    • "Before initiating an MSSV experiment, it is advisable to assess whether the proteins are spectrally distinguishable. In previous work (Padrick and Brautigam, 2011), we have established a numerical criterion (D norm ) that indicates whether the experimenter can reasonably expect a successful outcome from an MSSV experiment. For a two-component system in which the amino-acid compositions of both components are known and assuming the use of both ABS and IF, "
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    ABSTRACT: Modern computational strategies have allowed for the direct modeling of the sedimentation process of heterogeneous mixtures, resulting in sedimentation velocity (SV) size-distribution analyses with significantly improved detection limits and strongly enhanced resolution. These advances have transformed the practice of SV, rendering it the primary method of choice for most existing applications of analytical ultracentrifugation (AUC), such as the study of protein self- and hetero-association, the study of membrane proteins, and applications in biotechnology. New global multisignal modeling and mass conservation approaches in SV and sedimentation equilibrium (SE), in conjunction with the effective-particle framework for interpreting the sedimentation boundary structure of interacting systems, as well as tools for explicit modeling of the reaction/diffusion/sedimentation equations to experimental data, have led to more robust and more powerful strategies for the study of reversible protein interactions and multiprotein complexes. Furthermore, modern mathematical modeling capabilities have allowed for a detailed description of many experimental aspects of the acquired data, thus enabling novel experimental opportunities, with important implications for both sample preparation and data acquisition. The goal of the current unit is to describe the current tools for the study of soluble proteins, detergent-solubilized membrane proteins and their interactions by SV and SE. Curr. Protoc. Protein Sci. 71:20.12.1-20.12.49. © 2013 by John Wiley & Sons, Inc.
    Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 02/2013; Chapter 20:Unit20.12. DOI:10.1002/0471140864.ps2012s71
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    ABSTRACT: Sedimentation velocity (SV) experiments of heterogeneous interacting systems exhibit characteristic boundary structures that can usually be very easily recognized and quantified. For slowly interacting systems, the boundaries represent concentrations of macromolecular species sedimenting at different rates, and they can be interpreted directly with population models based solely on the mass action law. For fast reactions, migration and chemical reactions are coupled, and different, but equally easily discernable boundary structures appear. However, these features have not been commonly utilized for data analysis, for the lack of an intuitive and computationally simple model. The recently introduced effective particle theory (EPT) provides a suitable framework. Here, we review the motivation and theoretical basis of EPT, and explore practical aspects for its application. We introduce an EPT-based design tool for SV experiments of heterogeneous interactions in the software SEDPHAT. As a practical tool for the first step of data analysis, we describe how the boundary resolution of the sedimentation coefficient distribution c(s) can be further improved with a Bayesian adjustment of maximum entropy regularization to the case of heterogeneous interactions between molecules that have been previously studied separately. This can facilitate extracting the characteristic boundary features by integration of c(s). In a second step, these are assembled into isotherms as a function of total loading concentrations and fitted with EPT. Methods for addressing concentration errors in isotherms are discussed. Finally, in an experimental model system of alpha-chymotrypsin interacting with soybean trypsin inhibitor, we show that EPT provides an excellent description of the experimental sedimentation boundary structure of fast interacting systems.
    Methods 02/2011; 54(1):16-30. DOI:10.1016/j.ymeth.2011.01.010 · 3.65 Impact Factor
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    ABSTRACT: Gleaning information regarding the molecular physiology of macromolecular complexes requires knowledge of their component stoichiometries. In this work, a relatively new means of analyzing sedimentation velocity (SV) data from the analytical ultracentrifuge is examined in detail. The method depends on collecting concentration profile data simultaneously using multiple signals, like Rayleigh interferometry and UV spectrophotometry. If the cosedimenting components of a complex are spectrally distinguishable, continuous sedimentation-coefficient distributions specific for each component can be calculated to reveal the molar ratio of the complex's components. When combined with the hydrodynamic information available from the SV data, a stoichiometry can be derived. Herein, the spectral properties of sedimenting species are systematically explored to arrive at a predictive test for whether a set of macromolecules can be spectrally resolved in a multisignal SV (MSSV) experiment. Also, a graphical means of experimental design and criteria to judge the success of the spectral discrimination in MSSV are introduced. A detailed example of the analysis of MSSV experiments is offered, and the possibility of deriving equilibrium association constants from MSSV analyses is explored. Finally, successful implementations of MSSV are reviewed.
    Methods 05/2011; 54(1):39-55. DOI:10.1016/j.ymeth.2011.01.002 · 3.65 Impact Factor
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