Research Interests

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: * NSF CAREER AWARD   * University of California * Pittsburgh Supercomputer Center and Teragrid

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 (4MB wmv format)

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.

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.
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.
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.
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.
Ion channel selectivity and blocking mechanisms In collaboration with Crina Nimigean (Cornell) we have been investigating mechanisms of ion selectivity and blocking in potassium channels using electrophysiology and MD simulation. The highly conserved pore region of these channels is very selective for potassium, due to a series of backbone carbonyls projecting into the ion permeation pathway. Dehydration of smaller monovalent cations, such as sodium and lithium, is, however, highly unfavorable. Experimental studies reveal blocking by these smaller ions, yet also reveal interesting features at higher membrane voltages.
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.
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.	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 Download
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 (6MB mpeg format)
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.
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.