Properties of the free energy barriers for folding of the [alpha]-amylase inhibitor tendamistat

The goal of this work was to improve our understanding of the properties of the free
energy barriers in protein folding. We used the small all-[beta]-sheet protein tendamistat as
a model protein. Tendamistat contains two disulfide bridges and folds and unfolds in
apparent two-state reactions. However, previous studies on disulfide variants
demonstrated that tendamistat folds in at least two sequential steps with a high-energy
intermediate. Elucidation of the properties of the free energy barriers by studying
different tendamistat variants and various fragments provide a good insight into the
underlying complexity of apparent two state folders. To obtain this information several
approaches have been used in this work: we studied the combined influence of
denaturant, temperature, structural variation and sodium sulfate on folding and
stability; furthermore, we analysed the properties and stability of different fragments of
tendamistat.
Multiple perturbation analysis was used to gain information on the shape of the free
energy barriers in tendamistat folding. Analysis of denaturant and temperature as
perturbations revealed transition state movement according to the Hammond postulate.
Hammond behaviour is more pronounced in the early transition state compared to the
late transition state where only small transition state movement was observed. The
results suggest that the early transition state is rather broad compared to the late
transition state. These results emphasized the importance of multiple perturbation
analysis to test the shape of the free energy barriers in protein folding.
Determination of the activation parameters revealed less difference between both
transition states. However, the denaturant dependence of the activation parameters of
the transition states differs significantly. The results confirm our previous suggestion
that the early transition state is broad and structurally less well defined, whereas the
late transition state shows a rather narrow and structurally well-defined maximum.
We further studied the effect of denaturant and structural variation on folding and
stability. The results confirmed Hammond behaviour of the early transition state. To
know more about structural properties of the transition states we determined