Biomedical Engineering Reference
In-Depth Information
components. An ensemble of 15 structures was calculated from the
paramagnetic restraints which provided insights on RNA binding site
recognition and revealed novel interactions directly between the proteins.
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6.3.2.2 Protein-Protein Interactions
Protein association can be thought of as a two-step process in which a
transient, non-specific encounter complex precedes the formation of a well-
defined state.
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This encounter complex accelerates molecular recognition by
reducing the dimensionality of the conformational search.
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Until recently, the
study of these complexes was limited to theoretical models as they are invisible
to conventional techniques.
In 2003, Hansen et al. showed that PRE could be used to observe transient
protein complexes.
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However, it was not until 2006 that the first structural
ensembles representing invisible states were generated from PRE data using
two different biological systems. Initially, PRE was applied by Tang et al. to
the first complex of the bacterial phosphotransferase system between the N-
terminal domain of enzyme I (EIN) and the histidine phosphocarrier protein
(HPr). A Mn
2+
-chelating tag was attached, at three sites, to HPr and the inter-
and intramolecular PREs were recorded.
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It was found that neither the
specific complex, involving a pentacoordinate phosphoryl transition state
intermediate,
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nor any other single conformation could explain the observed
PRE profiles. Therefore, rigid-body simulated annealing was used to generate
an ensemble of structures representing an encounter complex that could fit the
PRE data. The ensemble contained 10-20 structures and accounted for 10% of
the population. Similar results were also shown for other phosphotransferase
complexes: IIA
mannitol
-HPr and IIA
mannose
-HPr.
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The work on the EIN-HPr complex was extended in 2007 by Suh et al. who
examined the dependence of intermolecular PRE on ionic strength. A Mn
2+
-
chelating tag was attached to EIN at two sites near the EIN-HPr interface.
The intermolecular PREs were significantly decreased in the presence of high
NaCl concentrations, suggesting that the formation of encounter complexes is
driven by electrostatic interactions.
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This work was extended further in 2010
by Fawzi et al. who showed that observing the R
2,para
on EIN as a function of
the concentration of paramagnetically labelled HPr allowed for identification
of two structurally distinct encounter complexes.
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Simultaneously in 2006, PRE was being applied by Volkov et al. to the
electron-transfer complex between yeast cytochrome c peroxidase (CcP) and
iso-1-cytochome c (Cc). Nitroxide spin-labels were attached at five locations
on the peroxidase surface and distance restraints were calculated from thePRE
data. Ensemble modelling showed that the major state was very similar to that
of the crystal structure and accounted for .70% of the lifetime of the complex.
The remainder of the lifetime was spent in a dynamic encounter complex.
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This work was continued in 2010 by Bashir et al. who combined rigid-body
Monte Carlo simulations, using only electrostatic and steric interactions, with
