Structural studies of membrane proteins have highlighted the likely influence of membrane mimetic environments (i.e., lipid bilayers versus detergent micelles) on the conformation and dynamics of small α-helical membrane proteins. We have used molecular dynamics simulations to compare the conformational dynamics of BM2 (a small α-helical protein from the membrane of influenza B) in a model phospholipid bilayer environment with its behavior in protein-detergent complexes with either the zwitterionic detergent dihexanoylphosphatidylcholine (DHPC) or the nonionic detergent dodecylmaltoside (DDM). We find that DDM more closely resembles the lipid bilayer in terms of its interaction with the protein, while the short-tailed DHPC molecule forms "nonphysiological" interactions with the protein termini. We find that the intrinsic micelle properties of each detergent are conserved upon formation of the protein-detergent complex. This implies that simulations of detergent micelles may be used to help select optimal conditions for experimental studies of membrane proteins.

α-Synuclein (αS) is a presynaptic protein that binds to cell membranes and is linked to Parkinson's disease (PD). Binding of αS to membranes is a likely first step in the molecular pathophysiology of PD. The αS molecule can adopt multiple conformations, being largely disordered in water, adopting a β-sheet conformation when present in amyloid fibrils, and forming a dynamic multiplicity of α-helical conformations when bound to lipid bilayers and related membrane-mimetic surfaces. Multiscale molecular dynamics simulations in conjunction with nuclear magnetic resonance (NMR) and cross-linking mass spectrometry (XLMS) measurements are used to explore the interactions of αS with an anionic lipid bilayer. The simulations and NMR measurements together reveal a break in the helical structure of the central non-amyloid-β component (NAC) region of αS in the vicinity of residues 65-70, which may facilitate subsequent oligomer formation. Coarse-grained simulations of αS starting from the structure of αS when bound to a detergent micelle reveal the overall pattern of protein contacts to anionic lipid bilayers, while subsequent all-atom simulations provide details of conformational changes upon membrane binding. In particular, simulations and NMR data for liposome-bound αS indicate incipient β-strand formation in the NAC region, which is supported by intramolecular contacts seen via XLMS and simulations. Markov state models based on the all-atom simulations suggest a mechanism of conformational change of membrane-bound αS via a dynamic helix break in the region of residue 65 in the NAC region. The emergent dynamic model of membrane-interacting αS advances our understanding of the mechanism of PD, potentially aiding the design of novel therapeutic approaches.

Potential of mean force (PMF) calculations are used to characterize the free energy landscape of protein-lipid and protein-protein association within membranes. Coarse-grained simulations allow binding free energies to be determined with reasonable statistical error. This accuracy relies on defining a good collective variable to describe the binding and unbinding transitions, and upon criteria for assessing the convergence of the simulation toward representative equilibrium sampling. As examples, we calculate protein-lipid binding PMFs for ANT/cardiolipin and Kir2.2/PIP2, using umbrella sampling on a distance coordinate. These highlight the importance of replica exchange between windows for convergence. The use of two independent sets of simulations, initiated from bound and unbound states, provide strong evidence for simulation convergence. For a model protein-protein interaction within a membrane, center-of-mass distance is shown to be a poor collective variable for describing transmembrane helix-helix dimerization. Instead, we employ an alternative intermolecular distance matrix RMS (DRMS) coordinate to obtain converged PMFs for the association of the glycophorin transmembrane domain. While the coarse-grained force field gives a reasonable Kd for dimerization, the majority of the bound population is revealed to be in a near-native conformation. Thus, the combination of a refined reaction coordinate with improved sampling reveals previously unnoticed complexities of the dimerization free energy landscape. We propose the use of replica-exchange umbrella sampling starting from different initial conditions as a robust approach for calculation of the binding energies in membrane simulations.

Lipid molecules can bind to specific sites on integral membrane proteins, modulating their structure and function. We have undertaken coarse-grained simulations to calculate free energy profiles for glycolipids and phospholipids interacting with modulatory sites on the transmembrane helix dimer of the EGF receptor within a lipid bilayer environment. We identify lipid interaction sites at each end of the transmembrane domain and compute interaction free energy profiles for lipids with these sites. Interaction free energies ranged from ca. -40 to -4 kJ/mol for different lipid species. Those lipids (glycolipid GM3 and phosphoinositide PIP2) known to modulate EGFR function exhibit the strongest binding to interaction sites on the EGFR, and we are able to reproduce the preference for interaction with GM3 over other glycolipids suggested by experiment. Mutation of amino acid residues essential for EGFR function reduce the binding free energy of these key lipid species. The residues interacting with the lipids in the simulations are in agreement with those suggested by experimental (mutational) studies. This approach provides a generalizable tool for characterizing the interactions of lipids that bind to specific sites on integral membrane proteins.