Non-Canonical Ligand-Binding Events as Detected by NMR - Recent Developments in Biomolecular NMR - page 237

The NMR titration data for the binding of Cd 2+ to this peptide is clearly
incompatible with simple two-site exchange. Several thermodynamic models can
be proposed that cause reduction of signal at supra-stoichiometric ratios. A
satisfactory model is high-affinity metal binding in the primary binding site in
species Q, followed by low-affinity binding in an auxiliary site. There is strong
evidence for (an) additional metal binding site(s), since the NOESY spectrum of
the peptide (further) changes when Cd 2+ is added in excess of stoichiometry. 21
Essential to the model from the perspective of the metal is, that additionalCd 2+
binding changes the properties of the primary binding site as shown in
Figure 10.11. If we choose the equilibrium binding constant K PQ 5 k PQ /k QP in
Figure 10.11 to be larger than K PR 5 k PR /k RP , than species Q will disappear
when the Cd 2+ concentration is increased in excess of stoichiometry. In the
process, species S will increase. In this model, one will observe the disappearance
of the signal with NMR properties when the metal concentration is increasedin
excess of stoichiometry.
As above, we used numerical integration to obtain the equilibrium
concentrations of the different species in the scheme of Figure 10.11. The
scheme was:

Figure 10.11

Modelling of two metal binding sites. The scheme has five
thermodynamic 'species', P, Q, R, S and free metal. The scheme
has five NMR species, free metal (a), b, c, d and e. The direct
communication between species Q and R ('Cd internalisation') is
important for the kinetics of metal binding but is thermodynamically
irrelevant. In the cartoons, Cd is a green circle, Cys-SH groups are
yellow circles, Glu-COO 2

groups are red circles and Lys NH 3 +

groups are blue circles.