concentration with time and radial position, are monitored in real
time using the optical system of the analytical ultracentrifuge.
Three optics are currently available, absorbance, interference, and
fluorescence optics which allow the detection of a variety of macromolecules in a broad range of concentrations. Compared to other
hydrodynamic techniques like dynamic light scattering (DLS) and
size exclusion chromatography, SV presents a higher hydrodynamic
resolution and a wider range of protein molecular weights to be
studied, from a few hundred daltons to several hundred-million
daltons, corresponding to peptides and large macromolecular complexes, respectively. Furthermore, the presence of contaminants or
aggregates below 1% can be quantified using SV [1, 2]. The analysis
of the sedimentation profiles allows the determination of the sedimentation and the diffusion coefficients of the macromolecules,
from the motion and shape of the sedimenting boundaries, and
the calculation of their molecular mass using the Svedberg equation
(see Subheading 1.1). For non-interacting species, these hydrodynamic parameters are directly related to the size and shape of
macromolecules. SV can also contribute to our understanding of
macromolecular assemblies with the determination of the equilibrium constant governing the interaction and the size of the macromolecular complex, and a robust approach to obtain these
parameters is the isotherm analysis. Binding isotherms are derived
from sedimentation coefficients distributions of a concentration
series and fit with a hetero-association model to obtain the dissociation constant and sedimentation coefficients of the macromolecular complexes. Concerning the stoichiometry of the complex, it is
deduced from the quality of the fit using different association
models but may require a more complicated analysis using multisignal SV approach, especially in the case of multi-component
systems. The dynamic range in binding affinities that can be
explored in SV is notably high from picomolar to millimolar K d
values due to the availability of the three optics and their different
sensitivity. It is noteworthy that the fluorescence detection system
that was recently developed allows now the study of protein interactions at low picomolar concentrations [3].
The present chapter aims to provide a theoretical background
of SV, to detail the SV protocols to characterize the heterogeneity
of a protein, and to measure a binary protein–protein interaction.
Two case studies will be presented. As SV technology and expertise
are accessible to researchers via many research infrastructures (see
Note 1), an additional goal of this chapter is to contribute to make
known the SV applications and raise interest for this technique.
More deep descriptions of the theory and applications of SV can
be found in recent reviews [2, 4–7].
156
Christine Ebel and Catherine Birck
time using the optical system of the analytical ultracentrifuge.
Three optics are currently available, absorbance, interference, and
fluorescence optics which allow the detection of a variety of macromolecules in a broad range of concentrations. Compared to other
hydrodynamic techniques like dynamic light scattering (DLS) and
size exclusion chromatography, SV presents a higher hydrodynamic
resolution and a wider range of protein molecular weights to be
studied, from a few hundred daltons to several hundred-million
daltons, corresponding to peptides and large macromolecular complexes, respectively. Furthermore, the presence of contaminants or
aggregates below 1% can be quantified using SV [1, 2]. The analysis
of the sedimentation profiles allows the determination of the sedimentation and the diffusion coefficients of the macromolecules,
from the motion and shape of the sedimenting boundaries, and
the calculation of their molecular mass using the Svedberg equation
(see Subheading 1.1). For non-interacting species, these hydrodynamic parameters are directly related to the size and shape of
macromolecules. SV can also contribute to our understanding of
macromolecular assemblies with the determination of the equilibrium constant governing the interaction and the size of the macromolecular complex, and a robust approach to obtain these
parameters is the isotherm analysis. Binding isotherms are derived
from sedimentation coefficients distributions of a concentration
series and fit with a hetero-association model to obtain the dissociation constant and sedimentation coefficients of the macromolecular complexes. Concerning the stoichiometry of the complex, it is
deduced from the quality of the fit using different association
models but may require a more complicated analysis using multisignal SV approach, especially in the case of multi-component
systems. The dynamic range in binding affinities that can be
explored in SV is notably high from picomolar to millimolar K d
values due to the availability of the three optics and their different
sensitivity. It is noteworthy that the fluorescence detection system
that was recently developed allows now the study of protein interactions at low picomolar concentrations [3].
The present chapter aims to provide a theoretical background
of SV, to detail the SV protocols to characterize the heterogeneity
of a protein, and to measure a binary protein–protein interaction.
Two case studies will be presented. As SV technology and expertise
are accessible to researchers via many research infrastructures (see
Note 1), an additional goal of this chapter is to contribute to make
known the SV applications and raise interest for this technique.
More deep descriptions of the theory and applications of SV can
be found in recent reviews [2, 4–7].
156
Christine Ebel and Catherine Birck