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Laboratory of Molecular Biophysics
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A number of ion channels contain transmembrane (TM) 
-helices which contain
proline-induced molecular hinges. These TM helices include the channel-forming
peptide alamethicin (Alm), the S6 helix from voltage-gated potassium (Kv)
channels, and the D5 helix from voltage-gated chloride (CLC) channels. The
hinge-bending motions of TM helices in bilayer-like environments have been
studied by multi-nanosecond MD simulations in an attempt to describe motions of
these helices that may underlie possible modes of channel gating. Alm is an
-helical channel-forming peptide, which contains a central kink associated
with a Gly-x-x-Pro motif in its sequence. Simulations of Alm in a TM
orientation for 10 ns in an octane slab indicate that the Gly-x-x-Pro motif
acts as a molecular hinge. A pattern matching approach was used to search for
possible hinge-bending motifs in the TM helices of other ion channel proteins.
This uncovered a conserved Gly-x-Pro motif in TM helix D5 of CLC channels. MD
simulations of a model of hCLC1-D5 spanning an octane slab suggest that this
channel also contains a TM helix that undergoes hinge-bending motion. These
simulations support a model in which hinge-bending motions of TM helices may
play a functional role in the gating mechanisms of several different families
of ion channels [8].
Previous MD simulations of isolated K channel helices [9] suggested that small distortions in pore lining helices (M2 in KcsA; S6 in Kv channels) might provide a mechanism for channel gating. Extended simulations of KcsA in a lipid bilayer suggest that a transient localised loss of helicity in just one of the four M2 helices is sufficient to 'open' the channel, enabling exit of a K+ ion from the central cavity. We are currently investigating the possible role of this mechanism in the physiological gating of the channel. In addition, simulation studies of the S6 helix from Kv channels (which contains a Pro-Val-Pro motif) have been extended. Simulations (10 ns) of an isolated KvS6 helix in an octane slab and in a POPC bilayer reveal hinge-bending motions, the frequency of which seems to be dependent on the fluidity of the membrane environment.
The structure of KcsA is almost certainly that of the closed state of the channel. It is known that certain tetra-alkyl ammonium ions (up to a carbon chain length of 5) can block via binding in the central cavity of this protein. For this to occur there must be some movement of the lower section of the protein in order to allow these ions to enter/exit to/from the central cavity. We are applying a number of non-equilibrium MD methods to explore how this movement may occur. Such techniques include "pulling" blockers out of the cavity with a spring, grand-canonical MC simulations to "force" additional waters in the cavity; and "growth" of an ever-expanding sphere at the gating mouth of the channel. By comparing results from several of these techniques to open the gate, it is envisaged that a common mechanism may become apparent.
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Proline induced helical kinks are not specific to Kv channels but are believed to occur in several channel and receptor proteins . This belief is supported by both experimental (e.g. from X-ray structures, fluorescence studies) and simulation data. In Kv channels the hinge corresponds to the Pro-X-Pro repeat in the S6 helix which produces a non-helical, flexible joint breaking the helix. This motif interestingly occurs in all Kv channels but is not found in KcsA. It is possible that a conformational change (produced for instance by communication with the voltage-sensing S4 helices) around the hinge in S6 may lead to the channel opening. Initial simulations of S6 in an octane slab will compare 10 simulations of 1ns duration and different starting conformation in order to improve sampling of possible S6 hinge conformations. In the longer term it is hoped to address the effect of mutations in the S6 helix on the hinge-bending dynamics.
Recent near atomic-resolution of the nicotinic acetylcholine receptor (N. Unwin, personal communication) suggest that even when this ion channel is functionally closed the pore appears to be physically open. The aim of the project is to test whether a short/narrow pore between two water-filled cavities can act as a gate, and how this behaviour depends on the length, radius and hydrophobicity of the pore. The pore is modelled as a cylinder made from pseudo-atoms which is embedded in an octane slab, mimicking the situation of a channel protein in the lipid bilayer of the membrane. We are employing classical molecular dynamics in order to describe the motion of water molecules within and around the pore. Initial simulations suggest that water molecules do not penetrate a purely hydrophobic pore with the dimensions of the narrowest region of the pore in the closed conformation of the nAChR.
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