mohammad tabish ahmed - eTheses Repository - University of ...

Chapter 5
it does support the idea that Cpn60.2 forms higher oligomeric structure in vivo, even if it is
very unstable. Then in chapter 4 it was shown that the precise apical domain swaps between
GroEL and Cpn60.2 resulted in functional proteins. This also supported the hypothesis that
Cpn60.2 functions using a similar, if not the same, mechanism than that of GroEL. It was
shown that both chimeras JNL and MKO were able to complement well in E. coli MGM100,
while the chimera AHC failed to do the same. What was interesting however was the
observation that, at least on native gels, JNL was a lower oligomer while AHC formed higher
oligomeric structures. However, since this observation was based entirely on data from native
gels, a better approach was needed.
For this reason, it was decided that JNL (Precise domain swap between Cpn60.2 and GroEL)
and AHC (imprecise domain swap between Cpn60.2 and GroEL) would be purified and the
protein samples would be analysed using analytical ultracentrifugation (AUC).
AUC is a technique that allows real time monitoring, during sedimentation, of the behaviours
of macromolecules in solution in their native state. This method allows the characterisation of
proteins by their hydrodynamic and thermodynamic properties in solution, without
interaction with any matrix or surfaces. There are two types of experiments that can be
performed with AUC, namely, sedimentation velocity and sedimentation equilibrium.
Sedimentation velocity is a hydrodynamic technique that measures the rates at which
molecules move in a centrifuge as a response to the centrifugal forces that are generated. This
method measures the entire time course of sedimentation and provides information on both
the shape and the molecular mass of the dissolved molecules. Sedimentation equilibrium on
the other hand is a thermodynamic technique that measures concentration distribution of
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