two) and yield more precise parameter estimates about K d , k off , and
s complex . This approach has been demonstrated to be efficient in a
number of cases but has several limitations including the fact that
individual species need to be reasonably described as discrete species with the correct molecular weights. This is not always the case,
for example, when impurities contribute to boundary broadening
and thus preclude a good model fitting and accurate results. Furthermore, as this method will take some time to converge, it is not
the first method of choice, but may be conducted using parameter
values obtained with the isotherm analysis.
1.4.2 Isotherm Analysis
to Characterize Protein
Hetero-association
For the construction of an isotherm, the first step is the c(s) analysis
of all SV data corresponding to the concentration series. In fact, the
c(s) analysis can also be applied to slowly as well as rapidly interacting systems. For slowly interacting systems, the boundary pattern
directly reflects the population of different species, which can often
be hydrodynamically resolved. In the case of a bimolecular interaction of the type A + B forming an AB complex with s-values such
that s A < s B < s AB , three boundaries will be observed corresponding
to free A, free B, and complex AB. For rapidly interacting systems,
the association-dissociation events during sedimentation lead to at
most two boundaries, one always sedimenting at the s-value of
either free A or free B, termed undisturbed boundary, and the
other at a composition-dependent s-value between s B and s AB ,
termed the reaction boundary. When the integration limits encompass all sedimenting species, the result is termed signal-weighted
average sedimentation coefficient, s w , which is directly related to the
overall transport of the interacting system and independent of the
kinetics of the interactions. When the integration limits encompass
only the reaction boundary, the result is termed s fast referring to the
sedimentation coefficient of the fast-sedimenting species. The
resulting s w and s fast isotherms can be combined and fitted with
mass action law models implemented in program SEDPHAT to
determine the binding constants and species size [19]. In addition
to these two s w and s fast isotherms, a population isotherm can be
constructed by integrating each c(s) peak and tabulating the
reported amplitudes, in signal units, and combined in the analysis.
1.4.3 Example of Protein
Hetero-association
We will use the association between the BTG2 factor and the poly
(A)-binding protein PABP C1 as model system to illustrate protein
hetero-association. These proteins were shown to interact directly
and their association sufficient in vitro and necessary in cellulo for
BTG2 to stimulate mRNA deadenylation. The BTG2/PABP interaction is also required for BTG2 to exert its antiproliferative
function [20].
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Christine Ebel and Catherine Birck
s complex . This approach has been demonstrated to be efficient in a
number of cases but has several limitations including the fact that
individual species need to be reasonably described as discrete species with the correct molecular weights. This is not always the case,
for example, when impurities contribute to boundary broadening
and thus preclude a good model fitting and accurate results. Furthermore, as this method will take some time to converge, it is not
the first method of choice, but may be conducted using parameter
values obtained with the isotherm analysis.
1.4.2 Isotherm Analysis
to Characterize Protein
Hetero-association
For the construction of an isotherm, the first step is the c(s) analysis
of all SV data corresponding to the concentration series. In fact, the
c(s) analysis can also be applied to slowly as well as rapidly interacting systems. For slowly interacting systems, the boundary pattern
directly reflects the population of different species, which can often
be hydrodynamically resolved. In the case of a bimolecular interaction of the type A + B forming an AB complex with s-values such
that s A < s B < s AB , three boundaries will be observed corresponding
to free A, free B, and complex AB. For rapidly interacting systems,
the association-dissociation events during sedimentation lead to at
most two boundaries, one always sedimenting at the s-value of
either free A or free B, termed undisturbed boundary, and the
other at a composition-dependent s-value between s B and s AB ,
termed the reaction boundary. When the integration limits encompass all sedimenting species, the result is termed signal-weighted
average sedimentation coefficient, s w , which is directly related to the
overall transport of the interacting system and independent of the
kinetics of the interactions. When the integration limits encompass
only the reaction boundary, the result is termed s fast referring to the
sedimentation coefficient of the fast-sedimenting species. The
resulting s w and s fast isotherms can be combined and fitted with
mass action law models implemented in program SEDPHAT to
determine the binding constants and species size [19]. In addition
to these two s w and s fast isotherms, a population isotherm can be
constructed by integrating each c(s) peak and tabulating the
reported amplitudes, in signal units, and combined in the analysis.
1.4.3 Example of Protein
Hetero-association
We will use the association between the BTG2 factor and the poly
(A)-binding protein PABP C1 as model system to illustrate protein
hetero-association. These proteins were shown to interact directly
and their association sufficient in vitro and necessary in cellulo for
BTG2 to stimulate mRNA deadenylation. The BTG2/PABP interaction is also required for BTG2 to exert its antiproliferative
function [20].
160
Christine Ebel and Catherine Birck