Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Deconvolution of Calcium-Binding Curves 35
of the parameters, experimental data obtained, either by measuring the number
of ligands bound to the protein or by following a spectroscopic signal as a
function of the ligand concentration, can be fitted to the model. One of the
main assumption of the model of Wang is the independence of the two lobes. If
this assumption is true, the behavior of whole calmodulin must be the sum of
the behaviors of its individual lobes (which can be obtained by proteolytic
digestion [51,57]). Some studies concluded that there was little or no interaction
between the N-terminal and C-terminal halves of calmodulin. This was
based on comparisons of titration curves of whole calmodulin vs those of isolated
domains (58–60). Other studies, using mutated calmodulins, were in
favor of an interaction between the two domains (61–64). A controversy arose
on this issue, as one cannot be sure that the tryptic fragments generated retain
their original structure, nor that mutagenesis does not induce minor changes in
the protein structure. It is only recently that work performed on whole
calmodulin set up his controversy and clearly established that the two lobes
of calmodulin were not independent (65–68). Also, in the presence of target
proteins or peptides, the cooperativity between the two lobes of CaM markedly
increases.
To get more insight into the mechanism of Ca 2+ -binding to calmodulin, we
decided in 1986 to build isofunctional mutants with an internal reporter group
(64,69,70) that will give us access to the occupancy of one specific site of the
protein. Our strategy was based on the reasoning developed in Subheading 3.
(see Fig. 2). As standard calmodulin does not harbor tryptophan residues in its
structure, we introduced one tryptophanyl residue at specific positions in
calmodulin (64). Results allowed us to confirm the model we proposed in 1981.
This model is presented in Fig. 3.
Each site exists in two different conformations: a conformation of high affinity
for Ca 2+ (HC conformation) and a low-affinity Ca 2+ -binding conformation
(LC conformation). In the apoform, only site III is in the HC conformation.
Upon Ca 2+ occupancy of this site, site IV undergoes a conformational change
toward a HC conformation, and so on, step by step, for the remaining sites. This
model is basically described by four constants, the individual binding constants
of each individual site in the HC conformation. If g1, g2, g3, and g4 are these
constants, the equation describing Ca 2+ -binding to calmodulin can be written
g 1 * (L) + 2 * g 1 * g 2 * (L) 2 + 3 * g 1 * g 2 * g 3 * (L) 3 + 4 * g 1 * g 2 * g 3 * g 4 (L) 4
v = —————————————————————————————— (18)
1 + g 1 * (L) + g 1 * g 2 * (L) 2 + g 1 * g 2 * g 3 * (L) 3 + g 1 * g 2 * g 3 * g 4 (L) 4
These constants (g i) correspond to the macroscopic constants of the Adair-Klotz
Eq. 2. In this model, we have a direct interpretation of the macroscopic constants
in terms of individual constants and coupling factors.