Phospholipid headgroups govern area per lipid and emergent elastic properties of bilayers.

Phospholipid bilayers are liquid-crystalline materials whose intermolecular interactions at mesoscopic length scales have key roles in the emergence of membrane physical properties. Here we investigated the combined effects of phospholipid polar headgroups and acyl chains on biophysical functions of membranes with solid-state 2H NMR spectroscopy. We compared the structural and dynamic properties of phosphatidylethanolamine and phosphatidylcholine with perdeuterated acyl chains in the solid-ordered (so) and liquid-disordered (ld) phases. Our analysis of spectral lineshapes of 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-d62) and 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphocholine (DPPC-d62) in the so (gel) phase indicated an all-trans rotating chain structure for both lipids. Greater segmental order parameters (SCD) were observed in the ld (liquid-crystalline) phase for DPPE-d62 than for DPPC-d62 membranes, while their mixtures had intermediate values irrespective of the deuterated lipid type. Our results suggest the SCD profiles of the acyl chains are governed by methylation of the headgroups and are averaged over the entire system. Variations in the acyl chain molecular dynamics were further investigated by spin-lattice (R1Z) and quadrupolar-order relaxation (R1Q) measurements. The two acyl-perdeuterated lipids showed distinct differences in relaxation behavior as a function of the order parameter. The R1Z rates had a square-law dependence on SCD, implying collective mesoscopic dynamics, with a higher bending rigidity for DPPE-d62 than for DPPC-d62 lipids. Remodeling of lipid average and dynamic properties by methylation of the headgroups thus provides a mechanism to control the actions of peptides and proteins in biomembranes.

How cholesterol stiffens unsaturated lipid membranes.

Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it is well understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure-property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach-combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (2H NMR) spectroscopy, and molecular dynamics (MD) simulations-we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer's packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure-property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol's role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid-protein interactions.

Nicotinamide phosphoribosyltransferase purification using SUMO expression system.

Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the salvage pathway required for nicotinamide adenine dinucleotide synthesis. The secreted NAMPT protein serves as a master regulatory cytokine involved in activation of evolutionarily conserved inflammatory networks. Appreciation of the role of NAMPT as a damage-associated molecular pattern protein (DAMP) has linked its activities to several disorders via Toll-like receptor 4 (TLR4) binding and inflammatory cascade activation. Information is currently lacking concerning the precise mode of the NAMPT protein functionality due to limited availability of purified protein for use in in vitro and in vivo studies. Here we report successful NAMPT expression using the pET-SUMO expression vector in E. coli strain SHuffle containing a hexa-His tag for purification. The Ulp1 protease was used to cleave the SUMO and hexa-His tags, and the protein was purified by immobilized-metal affinity chromatography. The protein yield was ~4 mg/L and initial biophysical characterization of the protein using circular dichroism revealed the secondary structural elements, while dynamic light scattering demonstrated the presence of oligomeric units. The NAMPT-SUMO showed a predominantly dimeric protein with functional enzymatic activity. Finally, we report NAMPT solubilization in n-dodecyl-ß-d-maltopyranoside (DDM) detergent in monomeric form, thus enhancing the opportunity for further structural and functional investigations.

Cholesterol Effects on the Physical Properties of Lipid Membranes Viewed by Solid-state NMR Spectroscopy.

In this chapter, we review the physical properties of lipid/cholesterol mixtures involving studies of model membranes using solid-state NMR spectroscopy. The approach allows one to quantify the average membrane structure, fluctuations, and elastic deformation upon cholesterol interaction. Emphasis is placed on understanding the membrane structural deformation and emergent fluctuations at an atomistic level. Lineshape measurements using solid-state NMR spectroscopy give equilibrium structural properties, while relaxation time measurements study the molecular dynamics over a wide timescale range. The equilibrium properties of glycerophospholipids, sphingolipids, and their binary and tertiary mixtures with cholesterol are accessible. Nonideal mixing of cholesterol with other lipids explains the occurrence of liquid-ordered domains. The entropic loss upon addition of cholesterol to sphingolipids is less than for glycerophospholipids, and may drive formation of lipid rafts. The functional dependence of 2H NMR spin-lattice relaxation (R 1Z) rates on segmental order parameters (S CD) for lipid membranes is indicative of emergent viscoelastic properties. Addition of cholesterol shows stiffening of the bilayer relative to the pure lipids and this effect is diminished for lanosterol. Opposite influences of cholesterol and detergents on collective dynamics and elasticity at an atomistic scale can potentially affect lipid raft formation in cellular membranes.

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.

Cholesterol-induced suppression of membrane elastic fluctuations at the atomistic level.

Applications of solid-state NMR spectroscopy for investigating the influences of lipid-cholesterol interactions on membrane fluctuations are reviewed in this paper. Emphasis is placed on understanding the energy landscapes and fluctuations at an emergent atomistic level. Solid-state (2)H NMR spectroscopy directly measures residual quadrupolar couplings (RQCs) due to individual C-(2)H labeled segments of the lipid molecules. Moreover, residual dipolar couplings (RDCs) of (13)C-(1)H bonds are obtained in separated local-field NMR spectroscopy. The distributions of RQC or RDC values give nearly complete profiles of the order parameters as a function of acyl segment position. Measured equilibrium properties of glycerophospholipids and sphingolipids including their binary and tertiary mixtures with cholesterol show unequal mixing associated with liquid-ordered domains. The entropic loss upon addition of cholesterol to sphingolipids is less than for glycerophospholipids and may drive the formation of lipid rafts. In addition relaxation time measurements enable one to study the molecular dynamics over a wide time-scale range. For (2)H NMR the experimental spin-lattice (R1Z) relaxation rates follow a theoretical square-law dependence on segmental order parameters (SCD) due to collective slow dynamics over mesoscopic length scales. The functional dependence for the liquid-crystalline lipid membranes is indicative of viscoelastic properties as they emerge from atomistic-level interactions. A striking decrease in square-law slope upon addition of cholesterol denotes stiffening relative to the pure lipid bilayers that is diminished in the case of lanosterol. Measured equilibrium properties and relaxation rates infer opposite influences of cholesterol and detergents on collective dynamics and elasticity at an atomistic scale that potentially affects lipid raft formation in cellular membranes.

Area per lipid and cholesterol interactions in membranes from separated local-field (13)C NMR spectroscopy.

Investigations of lipid membranes using NMR spectroscopy generally require isotopic labeling, often precluding structural studies of complex lipid systems. Solid-state (13)C 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 (13)C-(1)H 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 (13)C-(1)H 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 (13)C 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.

High-resolution NMR reveals secondary structure and folding of amino acid transporter from outer chloroplast membrane.

Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the (13)C, (15)N, (2)H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, HN, CO, Cα, and Cß chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T 1Z and T2 relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein.

Slow dynamics in a liquid crystal: 1H and 19F NMR relaxometry.

Spin-lattice relaxation rates (R(1H) and R(1F)) of two nuclear species ((1)H and (19)F) are measured at different temperatures in the isotropic phase of a liquid crystal (4(')-butoxy-3(')-fluoro-4-isothiocyanatotolane-4OFTOL), over a wide range of Larmor frequency (10 kHz-50 MHz). Their dispersion profiles are found to be qualitatively very different, and the R(1F) in particular shows significant dispersion (varying over two orders of magnitude) in the entire isotropic range, unlike R(1H). The proton spin-lattice relaxation, as has been established, is mediated by time modulation of magnetic dipolar interactions with other protons (case of like spins), and the discernable dispersion in the mid-frequency range, observed as the isotropic to nematic transition is approached on cooling, is indicative of the critical slowing of the time fluctuations of the nematic order. Significant dispersion seen in the R(1F) extending to very low frequencies suggests a distinctly different relaxation path which is exclusively sensitive to the ultra slow modes apparently present in the system. We find that under the conditions of our experiment at low Zeeman fields, spin-rotation coupling of the fluorine with the molecular angular momentum is the dominant mechanism, and the observed dispersion is thus attributed to the presence of slow torques experienced by the molecules, arising clearly from collective modes. Following the arguments advanced to explain similar slow processes inferred from earlier detailed ESR measurements in liquid crystals, we propose that slowly relaxing local structures representing such dynamic processes could be the likely underlying mechanism providing the necessary slow molecular angular momentum correlations to manifest as the observed low frequency dispersion. We also find that the effects of the onset of cross-relaxation between the two nuclear species when their resonance lines start overlapping at very low Larmor frequencies (below ~400 kHz), provide an additional relaxation contribution.