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Laboratory of Molecular Biophysics
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Previous: Viral Ion Channels, Next: Transport Proteins, Up: Ion Channels, Return to: Contents.
X-ray structures are known for a number of integral membrane proteins from the
outer membranes of Gram negative bacteria. These proteins are all based on a
-barrel architecture. Simulation studies are being used to explore the
relationship between protein structure and biological function for two classes
of such proteins.
FhuA, the receptor for ferrichrome-iron in E. coli, mediates the active transport of ferric siderophores across the outer membrane of Gram-negative bacteria. Crystallographic studies (reviewed in [14]) have indicated that ferrichrome-iron binding induces significant conformational changes in FhuA, in particular affecting the inner domain, known as the plug, and the periplasmic N-terminus, but the mechanism by which the transport takes place remains unclear. It has been proposed that these conformational changes may lead to the formation of a channel, wide enough to allow siderophore diffusion; a second model relies on a complete 'unplugging' of the N-terminal inner domain, which in turn would release the ligand in the periplasm, triggered by the interaction with of FhuA with TonB.
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During the last year MD simulations of FhuA embedded in a DMPC lipid bilayer
have been performed, in both its apo- and ferrichrome-bound states, in order to
give insight into the nature of this putative diffusion channel and the
conformational changes observed; the functional mutant FhuA
5-16 (from which
the N-terminal 'plug' domain has been deleted) has also been studied. These
simulations were run for up to 6 nanoseconds. Analysis of the simulation
results has focused on the structural stability and the flexibility of the
different domains of the protein, including the siderophore binding site, in
addition to both the intra-molecular and the protein-ligand hydrogen-bonding
patterns. The mobility of water and its association with the protein in the
barrel-cork interface have also been analysed.
The OmpA N-terminal fragment (residues 1-171, NTD) is an eight-stranded beta-barrel with no discernible pore through the protein, due to an extended hydrogen-bonded network within the barrel interior. Functional studies provide conflicting evidence as to whether OmpA plays a structural or ion-conducting (i.e. porin-like) role. Conductance estimates suggest that, when in an open state, OmpA may form a pore of diameter 3-10Å. To try to resolve this apparent paradox between functional and crystal structure data, a series of simulations on the OmpA-NTD have been undertaken. These include several ns-scale MD simulations, with both the crystal structure and a MC-solvated OmpA-NTD embedded in variable-dimension octane slabs, as well as in a DMPC bilayer.
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Initial results reveal that many of the crystal waters show a high degree of translational mobility; water molecules appear to be capable of crossing between most of the protein's internal cavities. The primary occlusion to pore activity is the Arg138-Glu52 salt bridge which appears to act as an effective barrier to water movement, at least on a ca. 1 ns timescale. However, the nearby Glu128 may be capable of providing an alternative ionic interaction with Arg138. Thus, it is conceivable that Arg138 may form alternative salt bridges with either of the two Glu side-chains, equivalent to closed and open states of an OmpA channel. The feasibility of this potential gating mechanism is being explored by MD simulations of an OmpA-NTD containing an alternative, restrained rotamer of Arg138.
Previous: Viral Ion Channels, Next: Transport Proteins, Up: Ion Channels, Return to: Contents.