Determination of protein complex stoichiometry through multisignal sedimentation velocity experiments.
Determination of the stoichiometry of macromolecular assemblies is fundamental to an understanding of how they function. Many different biophysical methodologies may be used to determine stoichiometry. In the past, both sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation have been employed to determine component stoichiometries. Recently, a method of globally analyzing multisignal sedimentation velocity data was introduced by Schuck and coworkers. This global analysis removes some of the experimental inconveniences and inaccuracies that could occur in the previously used strategies. This method uses spectral differences between the macromolecular components to decompose the well-known c(s) distribution into component distributions c(k)(s); that is, each component k has its own c(k)(s) distribution. Integration of these distributions allows the calculation of the populations of each component in cosedimenting complexes, yielding their stoichiometry. In our laboratories, we have used this method extensively to determine the component stoichiometries of several protein-protein complexes involved in cytoskeletal remodeling, sugar metabolism, and host-pathogen interactions. The overall method is described in detail in this work, as are experimental examples and caveats.
Padrick
Shae B
SB
Department of Biochemistry, University of Texas, Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
Deka
Ranjit K
RK
Chuang
Jacinta L
JL
Wynn
R Max
RM
Chuang
David T
DT
Norgard
Michael V
MV
Rosen
Michael K
MK
Brautigam
Chad A
CA
D051376:Actin-Related Protein 2-3 Complex / Q000737:chemistry; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry; D046912:Multiprotein Complexes / Q000737:chemistry*; D011485:Protein Binding; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry; D014461:Ultracentrifugation / Q000379:methods*
D051376:Actin-Related Protein 2-3 Complex; D046912:Multiprotein Complexes; D011768:Pyruvate Dehydrogenase Complex; D007781:Lactoferrin