Mean-torque Model Research Articles

Untangling the rivalry between cholesterol and antimicrobial peptides.

Antimicrobial peptides (AMPs) are promising candidates for next generation antimicrobial therapeutics. In mammalian cell membranes cholesterol plays a key regulatory function in antibiotic drug resistance and the immune response. However, the mechanism of action and the selectivity of AMPs towards bacterial membranes are not yet well understood. We hypothesized that differences in biophysical properties of mammalian versus bacterial membranes give rise to differences in peptide-membrane interactions which underlie the AMP mechanism and selectivity. Solid-state 2H NMR spectroscopy was employed to study di-monounsaturated phosphatidylcholine and phosphatidylethanolaminelipids (DOPC versus DOPE) bilayers in the presence of the antimicrobial peptide LL37 (2−4 mol%) and cholesterol (20−30% mol) as model systems mimicking mammalian and bacterial membranes. The lipid systems were probed with POPC-d31 (10 mol%) where the residual quadrupolar couplings yielded segmental order parameters (SCD) for the individual acyl segments. The LL37-peptide decreased the quadrupolar splittings indicating it increased the area per lipid, while cholesterol showed the opposite. The mean-torque model was used to calculate the key bilayer properties of area per lipid and bilayer thickness [2]. The model-free functional dependence of spin-lattice relaxation rates on SCD (square-law plots) indicated greater bending rigidity with cholesterol for both DOPC and DOPE membranes. By contrast LL37 decreased the bending rigidity of the membranes. Additionally all-atom molecular dynamics simulations for bilayers comprising POPE/DMPG/cardiolipin (90:5:5) and DMPC/cholesterol (90:10) were conducted with LL37 peptides on the bilayer surface to capture the molecular-level interactions. We observed partial insertion of the LL37-peptides in the POPE system, while the peptides were repelled from the membrane in the DMPC/cholesterol system. These observations reveal the surprising rivalry between cholesterol and AMPs which underlies AMP mechanism and selectivity.

Cholesterol promotes similar chain packing in n-3 PUFA-containing membranes.

Cholesterol's organization in biomembranes has great influence on membrane function. Omega-3 polyunsaturated fatty acid (n-3 PUFA)-containing lipids are believed to have a myriad of health benefits and are known to modulate membrane organization in model membranes via their aversion for cholesterol. 2H NMR has observed that different n-3 PUFA-containing phosphatidylcholine (PC) lipids modulate membrane organization to different degrees. In ternary lipid mixtures with sphingomyelin and cholesterol, docosahexaenoic acid (DHA, 22:6)-containing PC infiltrates lipid rafts more than eicosapentaenoic acid (EPA, 20:5)-containing PC. Additionally, molecular dynamics (MD) simulations have shown that n-3 PUFA-containing membranes differ in their stereospecific interactions with cholesterol. The mean-torque model provides a method of relating lipid disorder to a thermodynamic parameter (potential of mean torque) which can be used to quantify lipid-lipid interactions. Here we apply this approach to compare 1-stearoyl-2-eicosapentaenoylphosphatylcholine (EPA-PC, 18:0-20:5PC), 1-stearoyl-2-docosahexaenoylphosphatylcholine (DHA-PC, 18:0-22:6PC), 1-stearoyl-2-docosapentaenoylphosphatylcholine (DPA-PC, 18:0-22:5PC) and, as a monounsaturated control, 1-stearoyl-2-oleoylphosphatidylcholine (OA-PC, 18:0-18:1PC) bilayers and their interactions with cholesterol. Analysis of data from 2H NMR and MD simulations shows that all three n-3 PUFA-containing phospholipids display similar chain packing in the presence of cholesterol, which is illustrated by the projected chain length of the sn-1 chains.

Concepts and Methods of Solid-State NMR Spectroscopy Applied to Biomembranes.

Concepts of solid-state NMR spectroscopy and applications to fluid membranes are reviewed in this paper. Membrane lipids with 2H-labeled acyl chains or polar head groups are studied using 2H NMR to yield knowledge of their atomistic structures in relation to equilibrium properties. This review demonstrates the principles and applications of solid-state NMR by unifying dipolar and quadrupolar interactions and highlights the unique features offered by solid-state 2H NMR with experimental illustrations. For randomly oriented multilamellar lipids or aligned membranes, solid-state 2H NMR enables direct measurement of residual quadrupolar couplings (RQCs) due to individual C-2H-labeled segments. The distribution of RQC values gives nearly complete profiles of the segmental order parameters SCD(i) as a function of acyl segment position (i). Alternatively, one can measure residual dipolar couplings (RDCs) for natural abundance lipid samples to obtain segmental SCH order parameters. A theoretical mean-torque model provides acyl-packing profiles representing the cumulative chain extension along the normal to the aqueous interface. Equilibrium structural properties of fluid bilayers and various thermodynamic quantities can then be calculated, which describe the interactions with cholesterol, detergents, peptides, and integral membrane proteins and formation of lipid rafts. One can also obtain direct information for membrane-bound peptides or proteins by measuring RDCs using magic-angle spinning (MAS) in combination with dipolar recoupling methods. Solid-state NMR methods have been extensively applied to characterize model membranes and membrane-bound peptides and proteins, giving unique information on their conformations, orientations, and interactions in the natural liquid-crystalline state.

Elastic Deformation and Collective Dynamics in Lipid Membranes: A Solid-State 2H NMR Relaxation Study

Biomembrane functioning is significantly influenced by the composition and structure of the liquid-crystalline lipid bilayer [1]. Solid-state 2H NMR spectroscopy provides information about atomistic interactions among the membrane constituents by simultaneously probing structure and dynamics [2]. Here we examine the effect of water, osmolytes, cholesterol, and detergents on the liquid-crystalline properties of lipid membranes using NMR relaxation methods. We performed 2H NMR longitudinal (R1Z) and transverse quadrupolar-echo decay (R2QE) experiments on DMPC-d54 bilayers to study membrane lipid dynamics over the time scale ranging from nanoseconds to milliseconds. Plots of R1Z rates or transverse relaxation rates versus squared segmental order parameters (SCD2) show the emergence of collective lipid dynamics [3]. Such a functional behavior characterizes 3-D order-director fluctuations, due to the onset of membrane elasticity [3]. Yet at high hydration, a further R2QE enhancement and confinement of the functional square-law to the segments deeper in the bilayer is seen. Additional contributions from slower dynamics involving water-mediated membrane deformation are evident over mesoscopic length scales on the order of bilayer thickness. Such structural deformations are also evident from bilayer structural parameters calculated using a statistical mean-torque model [4]. The slow dynamics at high hydration or correspondingly low cholesterol or osmolyte concentration are due to modulation of elastic properties of the lipid bilayer. Analysis of the frequency dispersion of the transverse relaxation as a function of such external parameters reveals viscoelastic properties of the liquid-crystalline membranes. Such studies give insights into lipid rafts and membrane composition relevant for biomembrane function. [1] A. Leftin et al. (2014) Biophys. J. 107, 2274[2] K.J. Mallikarjuniah et al. (2011) Biophys. J.100, 98. [3] A. Leftin et al. (2014) eMagRes 4, 199. [4] J.J. Kinnun et al. (2015) BBA 1848, 246.

Area per Lipid and Cholesterol Interactions in Membranes from Separated Local-Field 13C NMR Spectroscopy

Investigations of lipid membranes using NMR spectroscopy generally require isotopic labeling, often precluding structural studies of complex lipid systems. Solid-state 13C magic-angle spinning NMR spectroscopy at natural isotopic abundance gives site-specific structural information that can aid in the characterization of complex biomembranes. Using the separated local-field experiment DROSS, we resolved 13C-1H residual dipolar couplings that were interpreted with a statistical mean-torque model. Liquid-disordered and liquid-ordered phases were characterized according to membrane thickness and average cross-sectional area per lipid. Knowledge of such structural parameters is vital for molecular dynamics simulations, and provides information about the balance of forces in membrane lipid bilayers. Experiments were conducted with both phosphatidylcholine (dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC)) and egg-yolk sphingomyelin (EYSM) lipids, and allowed us to extract segmental order parameters from the 13C-1H residual dipolar couplings. Order parameters were used to calculate membrane structural quantities, including the area per lipid and bilayer thickness. Relative to POPC, EYSM is more ordered in the ld phase and experiences less structural perturbation upon adding 50% cholesterol to form the lo phase. The loss of configurational entropy is smaller for EYSM than for POPC, thus favoring its interaction with cholesterol in raftlike lipid systems. Our studies show that solid-state 13C NMR spectroscopy is applicable to investigations of complex lipids and makes it possible to obtain structural parameters for biomembrane systems where isotope labeling may be prohibitive.

Area Per Lipid of Membranes from Natural Abundance Solid-State 13C NMR Spectroscopy

The properties of liquid-crystalline membranes vary according to the molecular composition of the lipid bilayer [1]. Structural investigations of lipid membranes using NMR spectroscopy generally require isotopic labeling of the lipids, thereby precluding investigations of complex lipid systems. Combining natural-abundance solid-state 13C magic-angle-spinning (MAS) NMR with a statistical mean torque (MT) model [2] enabled us to obtain the average area per lipid and volumetric bilayer thickness DB for lipid bilayers. The 2D separated local field (SLF) NMR experiment DROSS gives isotropic chemical shifts in the F2 frequency dimension and site-specific dipolar Pake doublets in the F1 dimension [1]. By analyzing the Pake patterns we extracted 13C-1H residual dipolar couplings (RDCs) and calculated segmental |SCH| order parameters. The |SCH| order parameters carry the structural information that aid calculation of and DB using the MT model. Experiments were conducted with DMPC, POPC, and EYSM lipids as single component (liquid-disordered, ld) lipid bilayers as well as with cholesterol. EYSM is more ordered in the ld phase and hence experiences less structural perturbation upon adding cholesterol to form the liquid-ordered (lo) phase relative to POPC. We infer that the cholesterol stiffening effect on lipid bilayers is limited by the maximum volumetric hydrocarbon thickness [2]. Knowledge of structural parameters like and DB is important for molecular dynamics (MD) simulations and provides information about the balance of forces in membrane lipid bilayers [3]. Our studies demonstrate the applicability of solid-state 13C NMR spectroscopy to site-specific structural investigations of complex lipids, where isotope labeling may be prohibitive, allowing structural studies of biologically relevant membrane systems. [1] A. Leftin et al.(2013) JMB425 2973-2987. [2] H. I. Petrache et al.(2000) BJ.79 3172-3192. [3] A. Leftin et al.(2011) BBA1808 818-839.

Experimental and Theoretical Comparison of Pressure Effects on Lipid Bilayer Fluctuations

Cell membrane fluctuations play crucial roles in membrane protein function and cell signaling, yet the specific biophysical mechanisms that control these fluctuations remain poorly understood. Solid-state 2H NMR is a powerful spectroscopic technique that has been used to quantify the free energy penalty of bilayer deformation, as well as the effect of increased osmotic pressure on bilayer properties including the surface area per lipid [1]. These experimental quantities are essential to force-field development and validation for the purpose of accurately modeling lipid bilayers at an atomistic or coarse-grained level. Our hypothesis is that a reformulation of the model used for calculation of segmental order parameters [2] will assist in examination and development of more accurate lipid force fields. This increase in accuracy will in turn lead to more realistic simulations that can provide invaluable insights about membrane bilayer behavior unattainable using other techniques. We carried out a series of concurrent solid-state 2H NMR and molecular dynamics (MD) studies on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers, a classical lipid model system, to examine the effect of applied surface tension (from −3 to 5 mJ m−2) on membrane behavior. For both experiment and simulation, as surface tension increased, the surface area per lipid decreased. Segmental order parameters were in general agreement between the two techniques. However, we found that MD simulations using the CHARMM c36 lipid force field tend to underestimate the increase in surface area per lipid with increased surface tension compared to 2H NMR-based results. Interestingly, the MD results match well with previous NMR studies that pre-date the mean-torque model used in our work, demonstrating the interplay between experiment and theory necessary for accurate force field development. [1] K.J. Mallikarjunaiah (2011) BJ 100, 98-107. [2] H.I. Petrache (2000) BJ 79, 3172-3192.

Investigation of synchroniser engagement in dual clutch transmission equipped powertrains

Transient response of a dual clutch transmission (DCT) powertrain to synchroniser mechanism engagements is investigated using a lumped inertia model of the powertrain. Original research integrates lumped inertia powertrain models for the DCT with a detailed synchroniser mechanism model and two separate engine models, comprising of a mean torque model and a harmonic torque model, using torque derived from piston firing. Simulations are used to investigate the synchroniser mechanism engagement process in a previously unscrutinised operating environment. Simulations are performed using both engine torque models, with the mean torque model demonstrates the highly nonlinear nature of synchroniser mechanism engagement, and the powertrain response to the engagement process. Through the introduction of harmonic engine torques, additional excitation is present in the mechanism during engagement, and increased vibration of the synchroniser sleeve results. The impact of vibrations is particularly important to the increased wear of indexing chamfer contact surfaces.

Raft-Like Phase Coexistence Revealed by Solid-State Carbon-13 Separated Local Field Mas NMR

Molecular interactions such as hydrogen-bonding and hydrophobic matching are observed for raft-like membranes using solid-state NMR [1]. We applied natural abundance separated-local field magic angle spinning 13C NMR to study the components of a canonical raft-like membrane system. This method probes lipid structure at the headgroup, glycerol backbone, and acyl chains simultaneously for each membrane component. We resolved isotropic 13C NMR chemical shifts and 13C-1H residual dipolar couplings (RDCs) for single-component, binary, and ternary membranes. These measurements are interpreted using a mean-torque model providing cross-sectional areas per lipid and volumetric hydrocarbon thicknesses [2]. The structural changes are related to mixing or de-mixing of lipids and cholesterol. In the single-component membranes, EYSM is highly ordered from hydrogen-bonding at the backbone, while POPC is highly disordered due to acyl chain unsaturation. The combination of these lipids leads to a reduction of EYSM order and an increase in POPC order due to inter-chain contacts. Addition of cholesterol yields a significant increase in hydrophobic matching in POPC and EYSM membranes [3]. From these structural results for single-component and binary systems, we evaluated the interactions contributing to lipid mixing in the ternary membrane system. EYSM exhibits a cross-sectional area and hydrocarbon thickness resembling the single-component system. In contrast, POPC exhibits a cross-sectional area and hydrocarbon thickness characteristic of the binary lipid-cholesterol mixture. This suggests a liquid-ordered phase is present in an ideally mixed ternary system. Our experimental results contribute to our understanding of raft-like membrane self-assembly, and can be used to interpret changes in membrane structure that occur during protein recognition events and membrane fusion. [1] T. Bartels et al. (2008) JACS 130 14521-14532. [2] H.I. Petrache et al. (2000) Biophys. J. 79, 3172-3192. [3] G.M. Martinez et al. (2004) Langmuir 20 1043-1046.