Biomedical Engineering Reference
In-Depth Information
Figure 10.14
Simulations of the five-site exchange NMR spectra according to the
model in Figures 10.11 and 10.13. Site a (free Cd(
II
)) resonates at
225 000 Hz; site b (the 'primary' buried site in species Q) at +25 000
Hz; site c (the surface site in species R) at 215 000 Hz ; site d (the
buried site in species S) at 0 Hz; site e (the surface site in species S) at
210 000 Hz. All sites have R
2
5 1000 s
21
. Only the signal for site b
(the buried site in species Q) at +25 000 Hz is shown. From bottom to
top: [Cd]
total
/ [P]
total
5 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2.00
with [P]
total
5 4 mM.
In summary, there are good reasons to propose that all Cd
2+
signals except
the one Cd
2+
in the symmetrical, stable binding site b in species Q are
unobservable due to exchange and CSA broadening effects.
The inferred presence of an external binding Cd
2+
site quite naturally leads
to a model of how an external binding site may be essential to the Cd
2+
binding
process to the central site. We envisage that Cd
2+
is initially coordinated by the
Glu residue(s) at the surface (red circles) located closest to the metal binding
Cys residues (yellow circles) in species R. As suggested by the data discussed
above, this leads to destabilisation of the Glu-Lys (blue circle) interaction
which in turn would facilitate the sequestration of the metal in the interior.
This model is attractive as it can account for relatively fast Cd
2+
binding to the
internal binding site as a two-step sequential process involving species Rand
Q. We believe that this model may also be of relevance for metal binding to
naturally occurring proteins.
In summary, what seemed to be a simple ligand-binding event according to
UV spectroscopy, emerged as a complicated five-site exchange mechanism as
deduced from very simple NMR experiments. The thermodynamic and kinetic
