Brett N. Olsen

Computational methods for biomolecular electrostatics.

An understanding of intermolecular interactions is essential for insight into how cells develop, operate, communicate, and control their activities. Such interactions include several components: contributions from linear, angular, and torsional forces in covalent bonds, van der waals forces, as well as electrostatics. Among the various components of molecular interactions, electrostatics are of special importance because of their long range and their influence on polar or charged molecules, including water, aqueous ions, and amino or nucleic acids, which are some of the primary components of living systems. Electrostatics, therefore, play important roles in determining the structure, motion, and function of a wide range of biological molecules. This chapter presents a brief overview of electrostatic interactions in cellular systems, with a particular focus on how computational tools can be used to investigate these types of interactions.

Perturbations of membrane structure by cholesterol and cholesterol derivatives are determined by sterol orientation

Cholesterol is essential for proper function and regulation of eukaryotic membranes, and significant amounts of metabolic energy are dedicated to controlling cellular cholesterol levels. Oxidation products of cholesterol, the oxysterols, are enzymatically produced molecules that play a major role in mediating cholesterol homeostasis through mechanisms which have not yet been fully elucidated. Certain oxysterols are known to have direct effects on membrane permeability and structure, effects that are strikingly different from that of cholesterol. We use molecular dynamics simulations of these oxysterols in 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) bilayers to explain the structural origins for the differing effects of cholesterol and 25-hydroxycholesterol on bilayer properties. In particular, we demonstrate that the source for these differing perturbations is the much wider range of molecular orientations accessible to 25-hydroxycholesterol when compared to cholesterol. This study shows that direct membrane perturbation by side-chain oxysterols is significant and suggests that these membrane perturbations may play a role in the oxysterol regulation of cholesterol homeostasis.

25-Hydroxycholesterol Increases the Availability of Cholesterol in Phospholipid Membranes

Side-chain oxysterols are enzymatically generated oxidation products of cholesterol that serve a central role in mediating cholesterol homeostasis. Recent work has shown that side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), alter membrane structure in very different ways from cholesterol, suggesting a possible mechanism for how these oxysterols regulate cholesterol homeostasis. Here we extend our previous work by using molecular-dynamics simulations of 25-HC and cholesterol mixtures in 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayers to examine the combined effects of 25-HC and cholesterol in the same bilayer. 25-HC causes larger changes in membrane structure when added to cholesterol-containing membranes than when added to cholesterol-free membranes. We also find that the presence of 25-HC changes the position, orientation, and solvent accessibility of cholesterol, shifting it into the water interface and thus increasing its availability to external acceptors. This is consistent with experimental results showing that oxysterols can trigger cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. These effects provide a potential mechanism for 25-HC-mediated regulation of cholesterol trafficking and homeostasis through modulation of cholesterol availability.

The Structural Basis of Cholesterol Accessibility in Membranes

Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity.

Improved Coarse-Grained Modeling of Cholesterol-Containing Lipid Bilayers.

Cholesterol trafficking, which is an essential function in mammalian cells, is intimately connected to molecular-scale interactions through cholesterol modulation of membrane structure and dynamics and interaction with membrane receptors. Since these effects of cholesterol occur on micro- to millisecond time scales, it is essential to develop accurate coarse-grained simulation models that can reach these time scales. Cholesterol has been shown experimentally to thicken the membrane and increase phospholipid tail order between 0 and 40% cholesterol, above which these effects plateau or slightly decrease. Here, we showed that the published MARTINI coarse-grained force-field for phospholipid (POPC) and cholesterol fails to capture these effects. Using reference atomistic simulations, we systematically modified POPC and cholesterol bonded parameters in MARTINI to improve its performance. We showed that the corrections to pseudobond angles between glycerol and the lipid tails and around the oleoyl double bond particle (the “angle-corrected model”) slightly improves the agreement of MARTINI with experimentally measured thermal, elastic, and dynamic properties of POPC membranes. The angle-corrected model improves prediction of the thickening and ordering effects up to 40% cholesterol but overestimates these effects at higher cholesterol concentration. In accordance with prior work that showed the cholesterol rough face methyl groups are important for limiting cholesterol self-association, we revised the coarse-grained representation of these methyl groups to better match cholesterol-cholesterol radial distribution functions from atomistic simulations. In addition, by using a finer-grained representation of the branched cholesterol tail than MARTINI, we improved predictions of lipid tail order and bilayer thickness across a wide range of concentrations. Finally, transferability testing shows that a model incorporating our revised parameters into DOPC outperforms other CG models in a DOPC/cholesterol simulation series, which further argues for its efficacy and generalizability. These results argue for the importance of systematic optimization for coarse-graining biologically important molecules like cholesterol with complicated molecular structure.

Characterization of Perfluorooctylbromide-Based Nanoemulsion Particles Using Atomistic Molecular Dynamics Simulations

Perfluorocarbon-based nanoemulsion particles have arisen as promising platforms for the cellular delivery of imaging and therapeutic agents to specific targets. However, current knowledge of the agent delivery mechanism is limited to qualitative and phenomenological models. Lack of detail at the molecular level has hence delayed optimizing or customizing nanoemulsion particles for therapeutic and imaging applications. Here we report the first atomistic structural details of a perfluorooctylbromide-based (PFOB-based) nanoemulsion particle (NEP) with a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) lipid emulsifier. Newly developed PFOB force-field parameters were used in molecular dynamics simulations to model the PFOB-NEP interface in a planar configuration. These PFOB force field parameters were developed and tested to reproduce the characteristics of bulk PFOB as well as PFOB at interfaces with water and emulsifying phospholipids. The modeled PFOB-NEP interface demonstrated significant intercalation of PFOB into the emulsifying lipid monolayer and consequent changes in the structural, electrostatic, and mechanical properties of the POPC monolayer and PFOB. This intercalation provides an explanation for experimental data demonstrating melittin tryptophan fluorescence quenching upon binding to the nanoemulsion particles through the observation of direct contact between the melittin tryptophan and the PFOB bromine. Additionally, the atomistic details of the PFOB-NEP interface structure provided by our simulations are used to suggest the influence of each component on PFOB-NEP delivery function which will be tested in future coarse-grained simulations.

Side-chain oxysterols modulate cholesterol accessibility through membrane remodeling.

Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), are key regulators of cholesterol homeostasis. New evidence suggests that the alteration of membrane structure by 25-HC contributes to its regulatory effects. We have examined the role of oxysterol membrane effects on cholesterol accessibility within the membrane using perfringolysin O (PFO), a cholesterol-dependent cytolysin that selectively binds accessible cholesterol, as a sensor of membrane cholesterol accessibility. We show that 25-HC increases cholesterol accessibility in a manner dependent on the membrane lipid composition. Structural analysis of molecular dynamics simulations reveals that increased cholesterol accessibility is associated with membrane thinning, and that the effects of 25-HC on cholesterol accessibility are driven by these changes in membrane thickness. Further, we find that the 25-HC antagonist LY295427 (agisterol) abrogates the membrane effects of 25-HC in a nonenantioselective manner, suggesting that agisterol antagonizes the cholesterol-homeostatic effects of 25-HC indirectly through its membrane interactions. These studies demonstrate that oxysterols regulate cholesterol accessibility, and thus the availability of cholesterol to be sensed and transported throughout the cell, by modulating the membrane environment. This work provides new insights into how alterations in membrane structure can be used to relay cholesterol regulatory signals.