Biomedical Engineering Reference
In-Depth Information
Figure 6.2
The ensemble of the Cc-Adx complexes from simulations based on RDC
data illustrates the intermolecular dynamics. Adx is shown as a blue
ribbon. The FeS cluster is shown as orange spheres. The geometrical
centers of Cc are represented by red spheres. The tensor frame of the
lanthanide tag for each conformation of Cc is represented as green sticks.
Reproduced with permission from ref.
43 . # Springer, 2009.
highly flexible domains that act as lids over the AMP and ATP binding sites.
By attaching a nitroxide spin-label to the AMP lid, severe line broadening was
observed for residues in the ATP lid. In the open state, these residues are too
far away from the spin-label to be affected by PRE suggesting that a partially
closed state is sampled in the absence of substrate. In combination with kinetic
data from RD and fluorescence microscopy, this sampling was shown not to be
random but instead to favor the catalytically competent closed state. 94
Together, these studies demonstrate the capability of PRE for analysing how
conformational selection aids in processes such as ligand binding.
6.3.2.4 Protein Folding
PRE is ideal for studying intrinsically disordered and unfolded proteins. 92 This
was done by Gillespie et al. in 1997 on the fragment model of the denatured
state of staphylococcal nuclease. Using nitroxide spin-labels, several long-
range interactions were identified that produced an ensemble topology very
similar to the topology of the native enzyme. 95,96 This suggested that
hydrophobic interactions may direct protein folding and since then many
other studies have used PRE, sometimes in combination with RDC, to identify
hydrophobic clusters in disordered proteins. 52,61,62,97-103
PRE has also been applied to intrinsically disordered proteins that cause
disease upon aggregation. In 2005, Bertoncini et al. and Dedmon et al. used
nitroxide spin-labels to study the topology of a-synuclein, which is involved in
Parkinson's disease. By converting the PRE data into distance restraints,
several long-range interactions were found that stabilised the native protein
under physiological conditions. In particular, the C-terminus was shown to
interact
with
the
hydrophobic
NAC
region.
These
interactions
prevent
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