Biomedical Engineering Reference
In-Depth Information
PRE data from nitroxide radicals attached at 10 sites on CcP. They found that
Cc sampled approximately 15% of the surface of CcP and established that the
encounter complex was indeed populated 30% of the time.
40
In 2010, Volkov et al. also used PRE data and Monte Carlo simulations to
show that the time spent in the Cc-CcP encounter complex could be
modulated. Three point mutations were made at the binding interface of Cc,
T12A, R13K and R13A, which changed the time spent in the encounter
complex from 30% in wild type to 10, 50 or 80%, respectively. The delicate
balance between the specific complex and the encounter complex was
explained as a consequence of biological function. The encounter complex
enhances the chance of forming a productive complex enormously, whereas the
specific complex is required for electron transfer. However, if the specific
complex were very stable, rapid turn-over would be hindered. Therefore, the
balance
between
the
two
is
a
compromise
that
meets
the
conflicting
requirements of rapid electron transfer and quick dissociation.
91
PRE has also been applied to other electron-transfer complexes including
the adrenodoxin (Adx)-Cc complex. In 2008, Xu et al. observed intermolecular
PCS and PREs, caused by the heme of Cc and the FeS cluster in Adx,
respectively, for an Adx-Cc complex that was cross-linked, but not for the
native complex. The lack of intermolecular paramagnetic effects for the native
complex was a result of averaging of multiple conformations of Cc, sampling
roughly 50% of the surface of Adx. This suggested that Adx-Cc exists solely as
an encounter complex.
42
This was confirmed by Xu et al. in 2009 using a rigid
lanthanide binding tag loaded with Yb
3+
and bound to Cc. As with the PCS
and PRE values of the previous study, a large drop in the RDC values was
observed when comparing the intramolecular (RDC on Cc) to the
intermolecular (RDC on Adx) values. Again, this was due to averaging,
which confirmed that the Adx-Cc complex is highly dynamic (Figure 6.2).
43
6.3.2.3 Protein Domain Rearrangements
Multi-domain proteins can undergo large conformational changes upon ligand
binding. Whether this shift from an 'open' to 'closed' state is entirely
dependent on the presence of ligand (induced fit) or if an equilibrium exists as
well for the ligand-free enzyme can be ascertained using PRE.
92
This was done
by Tang et al. in 2007 for the two-domain maltose binding protein. Using a
nitroxide spin-label, the ligand-free form of the protein was found to be in fast
exchange between the open and a partially closed state with the equilibrium
strongly favoring the open state (95%). Although ensemble modelling
generated a partially closed state that varied slightly from the ligand-bound
structure, the existence of such a transient structure suggested that conforma-
tional selection may play a role in maltose binding.
93
Similarly, in 2007, Henzler-Wildman et al. used PRE to study conforma-
tional changes in ligand-free adenylate kinase. Adenylate kinase catalyses the
reversible conversion of AMP and ATP to two ADP molecules. It has two
