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OCT 22, 2015 8:00 AM PDT

WEBINAR: Exploring the Stoichiometry of Macromolecular Complexes Using Multi-Signal Sedimentation Velocity Analytical Ultracentrifugation

Speaker
  • Associate Professor, Biophysics, Director, Macromolecular Biophysics Resource, The University of Texas Southwestern Medical Center
    Biography
      Chad A. Brautigam earned dual undergraduate degrees in Chemistry and Biochemistry and Molecular, Cellular, and Developmental Biology at the University of Colorado, Boulder. He then joined Thomas Steitz's lab at Yale University, where he studied the structural enzymology of DNA polymerases. Following the award of his Ph.D. in Molecular Biophysics and Biochemistry, Dr. Brautigam's postdoctoral work was in the laboratory of Johann Deisenhofer at the University of Texas Southwestern Medical Center. While in Dr. Deisenhofer's lab, he determined the three-dimensional structures of proteins involved in neuroplasticity (synapsins) and blue-light sensing in plants (cryptochromes). It was at this time that Dr. Brautigam first developed an interest in analytical ultracentrifugation (AUC). In 2007, he joined the faculty of UTSW, advancing to the rank of Associate Professor in the Department of Biophysics in 2012. Dr. Brautigam is also the director of the Macromolecular Biophysics Resource, a core laboratory offering training in and access to a variety of biophysical techniques. He has served as an instructor at AUC workshops around the world for over eight years. Additionally, Dr. Brautigam develops software for biophysical data processing, analysis, and presentation. These include REDATE and GUSSI, which both act on AUC data.

    Abstract
    A fundamental aspect of the thermodynamic characterization of macromolecular complexes is the determination of the component stoichiometry. There are a number of approaches that may be employed in this effort, including mass spectrometry, light scattering, and sedimentation equilibrium analytical ultracentrifugation (AUC). In recent years, an AUC method called multi-signal sedimentation velocity (MSSV) has emerged as an excellent means to elucidate stoichiometry. This technique, pioneered in the laboratory of Peter Schuck, capitalizes on the Beckman XL-A/I centrifuge’s ability to acquire sedimentation velocity (SV) data using multiple wavelengths and, in the case of the XL-I, multiple optical systems. Using the popular c(s) formalism, MSSV can supply the molar ratio of 2 – 4 species in a stable, co-sedimenting complex. In conjunction with the hydrodynamic information inherently available from a SV experiment, this often allows the unambiguous determination of stoichiometry. Basic theory and experimental design for MSSV will be featured in this webinar, along with the analytical strategies necessary to produce reliable results. Also, advanced topics, including measures that may be taken in difficult cases, will be covered. Throughout, concrete, real-world examples of MSSV will be presented.

    Learning Objectives:
    • Learn why an analytical ultracentrifugation (AUC) method called multi-signal sedimentation velocity (MSSV) has emerged as an excellent means to elucidate stoichiometry in recent years 
    • Learn the basic theory and experimental design for MSSV, along with the analytical strategies necessary to produce reliable results.
    • See real-world examples of MSSV application

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