Evaluating the stoichiometry of macromolecular complexes using multisignal sedimentation velocity

Shae B. Padrick, Chad A Brautigam

Research output: Contribution to journalReview article

20 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)39-55
Number of pages17
JournalMethods
Volume54
Issue number1
DOIs
StatePublished - May 1 2011

Keywords

  • Analytical ultracentrifugation
  • Arp2/3 complex
  • Biophysical methods
  • Multisignal sedimentation velocity
  • Step by step instructions
  • Stoichiometry

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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