Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

114 Lopez and Makhatadze
Fig. 1. The partial molar-heat capacity of ubiquitin at pH 2.95 obtained experimentally
(O) and fitted according to a two-state model (solid line), the partial molar heat
capacities of the native C p,N and unfolded states C p,U (dashed lines), the progress heat
capacity F N · C p,N + F U · C p,U (dotted line), the excess heat capacity C p exc experimental
(�) and fitted (solid line).
reality, sample and reference cells are slightly different and this difference has
to be taken into consideration by recording buffer/buffer scan. Thus prior to
starting the protein scan, it is very important to establish the stability of the
baseline, i.e., its relative position and shape.
One of the most important user-defined parameters is the heating rate. Several
considerations must be taken into account. First, the increase in sensitivity
is linear with respect to the heating rate, i.e., sensitivity with a heating rate
of 120°/h is twice higher than at 60°/h. One needs to keep in mind that the
increase in sensitivity actually leads to the decrease in the signal-to-noise ratio.
Second, if the expected transition is very sharp, e.g., occurs within a few
degrees, a high heating rate will distort the shape of the heat absorption profile
and lead to an error in the determination of all thermodynamic parameters
for this transition and, in particular, the transition temperature. Third,
the higher the heating rate, the less time the system has to relax to the equilibrium.
For slow unfolding/refolding processes, it is preferred to use low
heating rates. Usually small globular proteins exhibit fast folding/unfolding
and the heating rates of 90–120°/h are acceptable. For larger proteins, it is