Long Timescale Dynamics of Cholesterol in Lipid Bilayers
Total Page：16
File Type：pdf, Size：1020Kb
Long Timescale Dynamics of Cholesterol in Lipid Bilayers Proposal to the Biomedical Applications Group at the Pittsburgh Supercomputing Center, funded by the National Institute of General Medical Science June 23, 2016 Summary Cholesterol, the ubiquitous sterol present in mammalian cells, is a key player in maintaining and modifying characteristics of cell membranes, modulating the function of membrane-embedded and peripheral proteins. Cholesterol concentrations are cell type dependent, dynamic, and tightly regulated. Despite decades of experimental and computational studies, due to the fluidity and heterogeneity of the membrane environment, fundamental aspects of cholesterol function and dynamics are not yet understood. We propose to combine long timescale molecular dynamics with state-of-the-art magic-angle spinning solid-state NMR (SSNMR) experiments to yield an atomistically-resolved understanding of cholesterol. We have developed isotopic labeling and customized SSNMR measurements to provide site-resolved chemical shifts, order parameters and relaxation rates, which are experimental parameters dependent upon the hierarchical dynamics of the sterol in true lipid bilayers. These highly precise experimental data sets, when synergistically combined with computational analysis leveraging the unprecedented computational power of Anton 2, will enable a major breakthrough in understanding the molecular interactions of cholesterol in membranes. Our major goals are to use the long timescale MD simulations to discern for the first time the molecular interactions that lead to the hierarchical cholesterol-lipid and cholesterol-cholesterol dynamics. PI: Chad M. Rienstra Professor of Chemistry University of Illinois at Urbana-Champaign 600 South Mathews Avenue, Urbana, IL 61801 Telephone: (217) 244-4655 FAX: (217) 244-3186 Email: [email protected] Co-PI: Taras V. Pogorelov Research Assistant Professor of Chemistry Beckman Institute for Advanced Science and Technology National Center for Supercomputing Applications School of Chemical Sciences University of Illinois at Urbana-Champaign 600 South Mathews Avenue, Urbana, IL 61801 Telephone: (217) 244-4655 FAX: (217) 244-3186 Email: [email protected] 1 Background Sterols are vitally important in numerous biochemical and biophysical processes in the cell. Cholesterol, the major sterol present in mammalian cells, is a key regulator of membrane order, permeability, thickness and lateral organization1-3 and protein function1,4,5. Cholesterol also acts as a precursor to steroid hormones and bile acids6. The regulatory roles of cholesterol are postulated to depend directly upon interactions with other sterols and phospholipid molecules7-9. Recently it was shown that the orientation and dynamics of cholesterol are essential to its regulatory role10,11, though molecular details of the mechanism are still lacking. The importance of cholesterol to human health is appreciated by the fact that cholesterol-lowering drugs are a multibillion dollar per year global market, and almost 100 million Americans have high blood cholesterol (American Heart Association, 2013). Our research team is uniquely positioned to provide insights into this intense area of research by combining state-of-art solid-state NMR (SSNMR), biosynthesis and isotopic labeling, and molecular dynamics (MD) simulations to investigate the atomistically resolved hierarchical dynamics of cholesterol in lipid bilayers. Extensive prior studies of cholesterol have reported order parameters12 and restraints on the orientation in the bilayer from 2H SSNMR13,14. Additionally, there have been numerous MD simulations of cholesterol in the bilayer to ascertain the orientation and fast timescale dynamics (<100 ns)10,15. The limitations of these studies were the relatively small numbers of site- resolved experimental parameters and inadequate simulation timescales to examine biologically relevant events. We propose to rectify this situation in the current study and elucidate two fundamental aspects of cholesterol function: (1) a quantitative description of hierarchical dynamics and (2) cholesterol cluster formation and its influence on membrane. In both cases we plan to make extensive comparisons and analyses using both long timescale MD trajectories from Anton 2 and high-resolution 3D SSNMR data sets acquired at the University of Illinois. In our preliminary studies, we have generated foundational experimental data using 3D separated local field spectroscopy SSNMR methods in combination with highly 13C-enriched cholesterol samples prepared with an engineered yeast strain17. We thereby have determined site-specific chemical shifts and dipolar order parameters for cholesterol in multiple lipid bilayer environments above and below the gel-to-liquid-crystal phase transition, and we are in the process of measuring cross-correlated dipolar fields and relaxation parameters. These observables serve as high-resolution benchmarks that will be directly computed from the long timescale simulations to investigate hierarchical dynamics. The timescales of previous computational studies were too short to compute the important microsecond timescale motions involving clusters of cholesterol molecules. The unmatched computational power of Anton 2 (Ref. 18) will enable us to achieve an atomic-resolution description of cholesterol in phosphatidylcholine lipids with varying tail lengths to investigate the orientation, which depends upon lipid bilayer thickness10. The two lipid systems we propose to investigate are 10:3 POPC:Cholesterol and 10:3 DLPC:Cholesterol. These two lipid types, while having the same head-group, have a large difference in lipid tail length and membrane thickness. With the opportunity to capture long timescale simulations, we will be able to investigate the hierarchy of cholesterol’s diverse motions19: uniaxial molecular rotation, molecular axis wobble, chain reorientation and association to form cholesterol clusters (sometimes referred to as patches or rafts). Our data will lend insight into the distribution of size of these clusters, as well as their intermolecular association energetics and dynamics. Performing Anton 2 simulations at two different temperatures, corresponding to the liquid and gel phases, will distinguish between possible dynamic modes of cholesterol, that we are now able to capture with SSNMR. For example, the combination of experimental and computational data will distinguish among cholesterol rotating about its axis, rotating on the surface of a cone, and rotating and exploring the region within a cone. 2 Cholesterol is a vital part of the cell membrane and Anton 2 simulations proposed here when used in tandem with the unique SSNMR data will enable next level of understanding of the molecular mechanisms involved in regulation of the cell wall environment. Additionally, the direct comparison of SSNMR data with the long scale MD simulations will provide news insights into possible improvements of force fields. Scientific Objectives The Rienstra group has collected SSNMR data for most of the sites of cholesterol and now can connect experimental data directly with long timescale MD simulations. We have investigated cholesterol-lipid systems at two temperatures as these are conditions in which cholesterol is in environments both above and below the liquid-gel phase transition. Our SSNMR data indicate that substantial molecular motions remain even below the bulk phase transition temperature. In addition to the major fast (<100 ns) motional modes of uniaxial rotation and tail libration, slower motions (>microsecond) include translational diffusion, wobble on the surface of a cone, large amplitude tail reorientations and association events to form dimers and higher-order oligomers. Populations, rates, and Arrhenius activation energies for each of these processes can be extracted from the temperature-dependent SSNMR data for comparison with simulation. Our experimental results will complement the dynamic atomic-level description of how cholesterol interacts with the lipids. Anton 2 provides unique opportunities to model these systems on a long timescale to investigate both cholesterol-lipid and cholesterol-cholesterol interactions with experimental validation. We propose to simulate cholesterol-lipid systems (~120,000 atoms, Fig. 1) to fulfill our objectives. Effects of lipid tail length and temperature on cholesterol dynamics. To investigate how motions of cholesterol are influenced by the lipid tail length and hence membrane thickness, we will perform equilibrium simulations of the following cholesterol-lipid systems to match the SSNMR samples: 1) 10:3 POPC:Cholesterol and 2) 10:3 DLPC:Cholesterol. The former lipid tail has 16 and 18 carbon atoms while the latter has 12, and therefore the bilayer thickness differs and the orientation of cholesterol is altered dramatically. We anticipate fundamental changes in the interactions of cholesterol in each bilayer due to the altered Figure 1. (A) 13C-13C 2D Spectrum of cholesterol in POPC (B) Line-shape examples for C8 and C12, black is experimental and red is simulated compared the rigid CA of N-acetyl valine (NAV) (C) Cholesterol molecule with line-shape carbon sites highlighted and cholesterol depicted (purple) in POPC bilayers. 3 orientation and differences in available surface area for interaction with phospholipids. To match the conditions under which SSNMR data was collected and various dynamics modes were detected, we will perform simulations of each system at 37 °C and