Research Interests KcsA potassium ion channel (yellow ribbons) in a lipid membrane (gray/red hydrocarbon tails) in KCl (green/gray balls) solution.

Our research is aimed at applying theoretical and computational techniques to interesting biophysical problems. We're especially interested in the class of membrane proteins known as ion channels, which are essential for electrical and chemical activity in all organisms and responsible for many neurological and cardiovascular disorders. We aim to understand the microscopic driving forces of protein function in cell membrane environments. Supported by: * RMIT VC Senior Research Fellow  * RMIT Health Innovations Research Institute  * ARC Discovery Grant   * Chancellor's Fellow, UC Davis:p> * NSF Continuing Grant   * NCI Supercomputing facility   * VLSCI Supercomputing facility   * IVEC Supercomputing facility   * NSF Funded Teragrid   * National Resource for Biomedical Supercomputing

Ion channel selectivity/blocking Potassium channels allow K+ ions to diffuse through their pores while effectively blocking smaller Na+ ions: an ability that is vital for electrical and chemical activity in all organisms. This selection process is thought to arise because smaller ions do not bind in a thermodynamically favorable way. Using the KcsA channel, we are examining how Na+ and Li+ interact with the pore using MD simulations, as well as electrophysiology and X-ray crystallography with collaborator Crina Nimigean (Cornell). We have identified a binding site for Na+ and Li+ within the narrow selectivity filter. The position is such that the multi-ion permeation mechanism is disrupted leading to reduced ionic flux, without the ions ever entering the filter region. Download movie showing what K+ ions sees (Allen and Chung, 1999, 3MB wmv)

KcsA selectivity filter structure in the presence of Li ions (left) and proposed mechanisms of conduction (right) for different ions from the intracellular in the presence of K ions (red balls): K following path I and Na following path II with a big free energy barrier.

Thermodyamics of Ions and Ionizable groups in membranes Ionizable protein side chains play important roles in a wide array of biological phenomena, including: protein folding; protein-protein and protein-nucleic acid interactions; enzyme activity; pH activation of proteins; nuclear localization; peptide mediated membrane fusion; anti-microbial peptide activity; and various proton and ion transport mechanisms. The issue of ionizable protein side chains interacting with lipid membranes has been the focus of much attention since the proposal of the paddle model of voltage gated ion channels which suggested multiple arginine side chains may move through the hydrocarbon core of a lipid membrane, challenging a long standing view in membrane biophysics. We employ side chain analog and transmembrane helix models to determine the free energies of different ionizable protein side chains, as a function of protonation state, across membranes. Our findings have had implications for understanding the mechanisms of voltage gated ion channels and interpretations of translocon-based biological partitioning experiments. Download movie showing membrane deformations (Dorairaj and Allen, 2006, 4MB wmv) Download movie showing dipole potential distortion (Vorobyov and Allen, 2008, 4MB wmv)

An arginine side chain (gray/blue balls) on an alpha helix (green ribbon) traversing a membrane (gray tails, yellow P atoms), revealing the deformation of the lipids and water (red/white balls).

Ab initio, atomistic and coarse grained protein-lipid interactions Our membrane simulations have revealed that lipid bilayers will deform due to the presence of proteins, and especially charged solutes and protein side chains. This leads to a complex solvation microenvironment for which empirical force fields were not specifically parameterized. We have employed model systems to attempt to match experimental bulk partitioning and have carried out extensive quantum mechanical interaction calculations for protein-lipid interactions. Our findings demonatrate significant accuracy of free energy calculations emerging from both empirical-additive models as well as polarizable force field simulations. In contrast, recent implementations of coarse-grained simulations of protein-lipid interactions failed to reproduce the same structural changes in lipid bilayers, exhibit interactions that are an order of magnitude too small and subsequently lead to incorrect free energy profiles.

An arginine side chain analog (methyl-guanidinium) interacting with a lipid phosphate group analog (dimethyl-phosphate) in a cluster of water molecules to examine charged protein - membrane interactions.

Membrane Perturbations and Mismatch We have devised a series of projects employing simple transmembrane segments as models of membrane proteins, as used experimentally, to reveal general mechanisms of hydrophobic mismatch and its effects on protein stability and function. Changes in structure and dynamics of protein and lipids are examined and quantified thermodynamically by performing umbrella sampling and alchemical free-energy calculations.	A WALP (Trp-Ala-Leu-peptide; green ribbon with gray sticks for side chains) in a membrane (gray hydrocarbon with lipid P atoms as orange balls) undergoing response to hydrophobic mismatch.
Role of lipid composition on ion channel function In collaboration with Scott Feller (Wabash, Indiana) we have been simulating extensive trajectories of potassium channels to observe changes in protein structure, interactions, dynamics and gating-like vibrational modes in modified membranes. We have been examining the role of polyunsaturated lipids (e.g. omega 3, believed to promote cardiovascular and neurological health) which have been shown to affect G-protein-coupled-receptors in the retina and potassium channels in the brain and heart. Simulations reveal a close-packing of polyunsaturated lipids around the protein due to their greater flexibility.	KcsA channel (yellow ribbons) embedded in an SDPC membrane where DHA tails (red) preferentially solvate the transmembrane helices over stearic acid chains (gray).
Ionophore membrane permeation We are exploring the mechanisms of natural ionophore permeation across membranes using fully atomistic and continuum-based molecular dynamics simulations. For example, valinomycin is a neutral ionophore that is highly selective for potassium ions within lipid bilayers and functions as an potassium-specific transporter; but the microscopic details and thermodynamics underlying membrane permeability remain largely unknown.	Valinomycin with bound K ion (orange), as seen within the hydrophobic environment of the centre of a membrane.
Membrane protein conformational change Many membrane proteins, including ion channels, undergo conformational changes in transmembrane segments during activation. These changes will be modulated by the composition of the membrane, which consists of a bilayer arrangement of up to 200 different species of lipid, as well as amphiphilic molecules, proteins and pharmacological agents that can affect membrane mechanical and electrostatic properties. We are developing microscopic models to elucidate the general mechanical and electrostatic mechanisms of ion channel regulation in cell membranes by using free energy simulation methods to examine modulated protein conformational change. The ability to quantitatively predict and explain the effects of modifying agents on protein activity will represent a significant advance in computational biophysics.
Electrostatics of supported bilayers In collaboration with Atul Parikh (Applied Science) we have been exploring experimental and computational methods to quantify the asymmetric distribution of charged molecules between the leaflets of solid-substrate-supported phospholipid bilayers. On silica surfaces, negatively charged lipid components are shown to be enriched in the outer leaflet of a supported bilayer system at modest salt concentrations. The approaches developed provide a general means for exploring asymmetries of charged components in supported bilayer systems.	Experimental evidence of compositional heterogeneities in membranes supported on patterned surfaces (left), and theoretical calculations that predict charged lipid redistribution (right). For different concentrations of ions (50mM here), the energy difference of anionic lipids across a membrane, due to charged silica substrates, is shown.
Calcium binding proteins in vision In collaboration with Jim Ames (Chemistry), we are combining NMR and MD simulations to understand the function of the calcium sensor protein Recoverin involved in vision. We are currently using biased trajectory simulation methods to study the conformational changes of this protein upon calcium binding that lead to the protrusion of a Myr group that promotes membrane binding.	Model of recoverin binding to a membrane after calcium binding triggers extrusion of Myr switch (purple). Courtesy Jim Ames.
Ion channel permeation We have employed free energy techniques for understanding permeation and valence selectivity of narrow ion channels. We have explored permeation mechanisms by extracting multi-dimensional PMFs as a function of ion position, solvation and water alignment and have found semi-quantitative agreement with experimental conductance and binding measurements. We have been investigating the role of the protein in stabilizing different ions and the limitations of MD force-fields for modeling these extremely challenging process. Permeation GIF (Allen, 2004) Download Permeation Brownian Dynamics (Allen and Chung) Download (4MB wmv)	Top: Gramicidin A channel (yellow) sitting in a DMPC membrane (running vertical with gray hydrocarbon tails, in KCl solution (green K and gray Cl balls) with water in the channel shown as red/white balls). Bottom: Free energy surface for permeation.
Effects of protein thermal fluctuations on ion permeation: Atomic structures of proteins, including ion channels, usually are depicted as static entities. This provides for a convenient representation of the proteins overall structure, but at the risk of deemphasizing the importance of thermal fluctuations in the protein function.These fluctuations become particularly important in narrow pores. Descriptions of ion permeation which employ static protein structures and a macroscopic continuum dielectric solvent face significant difficulties. Download Movie Showing Fluctuations (Allen, 2004, 6MB mpeg)	Variation in energetics of ion permeation through gramicidin A for different configurtions of the thermally fluctuating protein, based on rigid protein Poisson-Boltzmann solutions.
MD as a tool for NMR protein structural refinement Solid-state NMR has become increasingly important for investigating the structure of membrane proteins. Microscopic models incorporating a realistic molecular environment can be used to generate a dynamic ensemble that reproduces experimental observations with remarkable precision. Our results demonstrate that MD is a powerful tool for membrane protein structural refinement.	Two different rotamers of a Trp on the gramicidin A channel from SS (1MAG) and solution (JNO) NMR. Simulations of the protein in membranes demonstrate that the SS NMR structure does not reproduce SS NMR data.
Methodological improvements Slow movement of lipid molecules in a bilayer poses a difficulty for investigating protein function. We're developing improved sampling techniques that combine umbrella sampling and Monte Carlo based methodologies.