A Mass-Spectrometry-Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins

Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non-specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid-binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent-resistant lipids bound at the dimer interface in the leucine transporter show decreased k_off rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid-II results in the formation of a 1:1 protein–lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non-annular lipids based on their exchange rates in solution.     

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry‐Based Approach

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three‐dimensional structures. As lipids are dynamic by nature, their “structure” does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid‐raft‐related molecules, lipid–protein interactions, and membrane‐active natural products, we discuss current perspectives on membrane structural biology.     

Fabrication of a Retinal‐protein Photoreceptor Array

A pixel‐architecture film of retinal proteins was prepared by an approach combining chemical, physical and biological technologies. Oriented multilayers of purple membrane composed of bacteriorhodopsin (BR) and lipids were patterned on an array of gold electrode pixels. In order to improve stability and resolution, the gene engineering technique was employed to make a mutant of the protein BR by replacing the 36^th amino acid residue from aspartic acid to cysteine with a thiol end group ready to react with gold; electric sedimentation was used to guarantee the high probability of formation of the Au‐S bond and meanwhile to orient BR; further chemical crosslinking was introduced among layers of purple membranes to significantly enhance photoelectrical signals while keeping high stability. The non‐bound BR region was eventually washed out by detergent, and the remaining BR pixels were thus detergent resistant due to chemical crosslinking among BR layers and covalent binding between the multilayer and the substrate. The protein array was confirmed to keep photoelectrical activity.     

Separation and characterization of oxidized isomeric lipid–peptide adducts by ion mobility mass spectrometry

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid–protein adducts by liquid chromatography‐MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid–peptide adducts generated by incubating model peptides corresponding to the amphipathic β₁ sheet sequence of apolipoprotein B‐100 with 1‐palmitoyl‐2‐(oxo‐nonanoyl)‐sn‐glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide–lipid adducts was separated by TWIMS, which was especially important for the identification of two mono‐PONPC‐peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide–lipid adduct possessing most likely different orientations of the hydrophobic sn‐1 fatty acyl residue and head group of PONPC, relative to the peptide backbone. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of lipid‐soluble arsenic species in seaweed‐eating sheep from Orkney

This work is part of an ongoing research study towards an understanding of the complete metabolism of arsenosugars in mammalian organisms when ingesting seaweed, using the North Ronaldsay (NR) sheep as a model organism. We focus on the analysis of only those arsenic species bound to the lipids of the feed (Laminaria digitata), faeces and the tissues of the NR sheep using a novel enzymatic hydrolytic method that is simple and reliable. This rare breed of sheep, found in the remote Orkney Islands in the north of Scotland, live the entire year on the beaches and eat seaweed that is washed ashore (up to 3 kg daily). Previous studies on arsenic fractionation in muscle, kidney and liver tissues revealed that most of the arsenic is concentrated in the fat fractions of these tissues (muscle fat: 61%; liver fat: 66%; kidney fat: 25%) rather than in the non‐lipid fractions. Hence, this study was undertaken in order to determine the arsenic species bound to lipids in the muscle, kidney and faeces of NR sheep and to compare these with the arsenic species bound to the lipids of the L. digitata consumed. The enzymatic hydrolytic procedure has been successfully employed for the first time to cleave the arsenic species cleanly from the rest of the lipid structure. This makes the arsenic species water soluble and enables their direct determination by high‐performance liquid chromatography coupled with inductively coupled plasma mass spectrometry. Dimethylarsinic acid (DMA(V)) and monomethylarsonic acid (MA(V)) were found to be the major hydrolysed arsenic species bound to the kidney and muscle lipids, whereas arsenosugar‐1 was found to be the major hydrolysed arsenic species in L. digitata lipids. On the other hand, DMA(V) was found to be the major arsenical obtained after the enzymatic hydrolysis of the faeces lipids. These results seem to suggest that both direct absorption and biotransformation of the absorbed organoarsenicals are the likely reasons for their occurrence and accumulation in the NR sheep tissues. Copyright © 2003 John Wiley & Sons, Ltd.     

Computational study of ligand binding in lipid transfer proteins: Structures,interfaces, and free energies of protein‐lipid complexes

Plant nonspecific lipid transfer proteins (nsLTPs) bind a wide variety of lipids, which allows them to perform disparate functions. Recent reports on their multifunctionality in plant growth processes have posed new questions on the versatile binding abilities of these proteins. The lack of binding specificity has been customarily explained in qualitative terms on the basis of a supposed structural flexibility and nonspecificity of hydrophobic protein‐ligand interactions. We present here a computational study of protein‐ligand complexes formed between five nsLTPs and seven lipids bound in two different ways in every receptor protein. After optimizing geometries in molecular dynamics calculations, we computed Poisson‐Boltzmann electrostatic potentials, solvation energies, properties of the protein‐ligand interfaces, and estimates of binding free energies of the resulting complexes. Our results provide the first quantitative information on the ligand abilities of nsLTPs, shed new light into protein‐lipid interactions, and reveal new features which supplement commonly held assumptions on their lack of binding specificity. © 2012 Wiley Periodicals, Inc.     

CHARMM‐GUI Membrane Builder toward realistic biological membrane simulations

CHARMM‐GUI Membrane Builder, http://www.charmm‐gui.org/input/membrane , is a web‐based user interface designed to interactively build all‐atom protein/membrane or membrane‐only systems for molecular dynamics simulations through an automated optimized process. In this work, we describe the new features and major improvements in Membrane Builder that allow users to robustly build realistic biological membrane systems, including (1) addition of new lipid types, such as phosphoinositides, cardiolipin (CL), sphingolipids, bacterial lipids, and ergosterol, yielding more than 180 lipid types, (2) enhanced building procedure for lipid packing around protein, (3) reliable algorithm to detect lipid tail penetration to ring structures and protein surface, (4) distance‐based algorithm for faster initial ion displacement, (5) CHARMM inputs for P₂₁ image transformation, and (6) NAMD equilibration and production inputs. The robustness of these new features is illustrated by building and simulating a membrane model of the polar and septal regions of E. coli membrane, which contains five lipid types: CL lipids with two types of acyl chains and phosphatidylethanolamine lipids with three types of acyl chains. It is our hope that CHARMM‐GUI Membrane Builder becomes a useful tool for simulation studies to better understand the structure and dynamics of proteins and lipids in realistic biological membrane environments. © 2014 Wiley Periodicals, Inc.     

Identification of a lipid‐related peak set to enhance the interpretation of TOF‐SIMS data from model and cellular membranes

Principal component analysis (PCA) of time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS) data enables differentiating structurally similar molecules according to linear combinations of multiple peaks in their spectra. However, in order to use PCA to correctly identify variations in lipid composition between samples, the discrimination achieved must be based on chemical differences that are related to the lipid species, and not sample‐associated contamination. Here, we identify the positive‐ion TOF‐SIMS peaks that are related to phosphatidylcholine lipid headgroups and tail groups by PCA of spectra acquired from lipid isotopologs. We demonstrate that restricting PCA to a contaminant‐free lipid‐related peak set reduces the variability in the spectra acquired from lipid samples that is due to contaminants, which enhanced differentiating different lipid standards, but adversely affected the contrast in PC scores images of phase‐separated lipid membranes. We also show that PCA of a restricted data set consisting of the peaks related to lipids and amino acids increases the likelihood that the discrimination of TOF‐SIMS data acquired from intact cells is based on differences in the lipids and proteins on the cell surface, and not sample‐specific contamination without compromising sample discrimination. We expect that the lipid‐related peak database established herein will facilitate interpreting the TOF‐SIMS data and PCA results from studies of both model and cellular membranes, and enhance identifying the origins of the peaks that contribute to discriminating different types of cells. Copyright © 2011 John Wiley & Sons, Ltd.     

Probing the Lipid Annular Belt by Gas‐Phase Dissociation of Membrane Proteins in Nanodiscs

Interactions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas‐phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein–lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment.     

Lateral Organization of Host Heterogeneous Raft‐like Membranes Altered by the Myristoyl Modification of Tyrosine Kinase c‐Src

Membrane‐bound c‐Src non‐receptor tyrosine kinase, unlike other acyl‐modified lipid‐anchored proteins, anchors to the membrane by a myristoyl chain along with a polybasic residue stretch, which is shorter in chain length than its host membrane. The packing defect arising from this mismatched chain length of the host and the lipid anchor significantly affects the lateral organization of heterogeneous membranes. We reveal the mixing of phase domains and formation of novel nanoscale‐clusters upon membrane binding of the Myr‐Src (2–9) peptide. Fluorescence cross correlation spectroscopy was used to explore the nature of these clusters. We show that Myr‐Src (2–9) is able to oligomerize, and the peptide clusters are embedded in a lipid platform generated by lipid sorting. Further, using confocal fluorescence microscopy and FRET assays we show that localized charge enrichment and membrane curvature are able to shift the partition coefficient towards the more ordered lipid phase.     

Self‐Assembled Aggregates and Molecular Bilayer Films of a Double‐Chain Fullerene Lipid: Structure and Electrochemistry

The self‐aggregation behavior of C₆₀ fullerenes that bear two octadecyl chains (lipid 1 ) as well as the structures and electrochemical properties of cast films of 1 are described. We also examined the self‐aggregation behavior in organic solvents of three previously reported compounds: C₆₀ with three each of hexadecyl (lipid 2 ), tetradecyl (lipid 3 ), or dodecyl (lipid 4 ) chains. The fullerene lipids in alcohols spontaneously formed spherical aggregates, whose diameters are related to the alkyl‐chain lengths, concentrations of the fullerene lipids, and the solvent polarity. The morphologies of the aggregates showed temperature dependence. Cast films of 1 formed multimolecular bilayer structures that undergo a phase transition typical of lipid bilayer membranes. The electrochemistry of cast films of 1 on an electrode in aqueous medium exhibits temperature dependence.     

Dihedral‐Angle‐Controlled Crossover from Static Hole Delocalization to Dynamic Hopping in Biaryl Cation Radicals

In cases of coherent charge‐transfer mechanism in biaryl compounds the rates follow a squared cosine trend with varying dihedral angle. Herein we demonstrate using a series of biaryl cation radicals with varying dihedral angles that the hole stabilization shows two different regimes where the mechanism of the hole stabilization switches over from (static) delocalization over both aryl rings to (dynamic) hopping. The experimental data and DFT calculations of biaryls with different dihedral angles unequivocally support that a crossover from delocalization to hopping occurs at a unique dihedral angle where the electronic coupling (H _ab) is one half of reorganization (λ ), that is, H _ab=λ /2. The implication of this finding in non‐coherent charge‐transfer rates is being investigated.     

Site‐Resolved Observation of Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater

Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in a number of theoretical studies. Experimental evidence for such pathways, however, is sparse because site‐selective injection of vibrational energy into a protein, that is, localized heating, is required for their investigation. Here, we solved this problem by the site‐specific incorporation of the non‐canonical amino acid β‐(1‐azulenyl)‐l ‐alanine (AzAla) through genetic code expansion. As an exception to Kasha's rule, AzAla undergoes ultrafast internal conversion and heating after S₁ excitation while upon S₂ excitation, it serves as a fluorescent label. We equipped PDZ3, a protein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions. We indeed observed VET from the incorporation sites in the protein to a bound peptide ligand on the picosecond timescale by ultrafast IR spectroscopy. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role in a wide range of proteins.     

Effect of Headgroups on Small‐Ion Permeability across Archaea‐Inspired Tetraether Lipid Membranes

This paper examines the effects of four different polar headgroups on small‐ion membrane permeability from liposomes comprised of Archaea‐inspired glycerolmonoalkyl glycerol tetraether (GMGT) lipids. We found that the membrane‐leakage rate across GMGT lipid membranes varied by a factor of ≤1.6 as a function of headgroup structure. However, the leakage rates of small ions across membranes comprised of commercial bilayer‐forming 1‐palmitoyl‐2‐oleoyl‐sn‐glycerol (PO) lipids varied by as much as 32‐fold within the same series of headgroups. These results demonstrate that membrane leakage from GMGT lipids is less influenced by headgroup structure, making it possible to tailor the structure of the polar headgroups on GMGT lipids while retaining predictable leakage properties of membranes comprised of these tethered lipids.     

Self‐Consistent Field Modeling of Three‐Dimensional Morphologies of Branched Lipid Surfactant at Air‐Water Interface

Three‐dimensional mesoscopic morphologies and the thermodynamics of structural phase transitions of amphiphilic lipids at air‐water interfaces are studied using self‐consistent field theory. Changing the relative amount of lipids in the system led to a series of 3D morphologic phases with varying average interfacial area per molecule, mimicking a compression of the model membranes. Membranes of both saturated and unsaturated lipids undergo a transition from cylindrical micelle to lamella when the lipid content in the system increases from 2% to about 19–20%. With further increase in the lipid content, saturated lipids first develop non‐uniform quasi‐2D distributions in the lamella and then gradually transform into a hybrid morphology containing quasi‐planar lamellae. In contrast, unsaturated lipids develop reverse‐micellar morphologies.

     

Cyclohexane Rings Reduce Membrane Permeability to Small Ions in Archaea‐Inspired Tetraether Lipids

Extremophile archaeal organisms overcome problems of membrane permeability by producing lipids with structural elements that putatively improve membrane integrity compared to lipids from other life forms. Herein, we describe a series of lipids that mimic some key structural features of archaeal lipids, such as: 1) single tethering of lipid tails to create fully transmembrane tetraether lipids and 2) the incorporation of small rings into these tethered segments. We found that membranes formed from pure tetraether lipids leaked small ions at a rate that was about two orders of magnitude slower than common bilayer‐forming lipids. Incorporation of cyclopentane rings into the tetraether lipids did not affect membrane leakage, whereas a cyclohexane ring reduced leakage by an additional 40 %. These results show that mimicking certain structural features of natural archaeal lipids results in improved membrane integrity, which may help overcome limitations of many current lipid‐based technologies.     

Bioinspired,Size‐Tunable Self‐Assembly of Polymer–Lipid Bilayer Nanodiscs

Polymer‐based nanodiscs are valuable tools in biomedical research that can offer a detergent‐free solubilization of membrane proteins maintaining their native lipid environment. Herein, we introduce a novel ca. 1.6 kDa SMA‐based polymer with styrene:maleic acid moieties that can form nanodiscs containing a planar lipid bilayer which are useful to reconstitute membrane proteins for structural and functional studies. The physicochemical properties and the mechanism of formation of polymer‐based nanodiscs are characterized by light scattering, NMR, FT‐IR, and TEM. A remarkable feature is that nanodiscs of different sizes, from nanometer to sub‐micrometer diameter, can be produced by varying the lipid‐to‐polymer ratio. The small‐size nanodiscs (up to ca. 30 nm diameter) can be used for solution NMR spectroscopy studies whereas the magnetic‐alignment of macro‐nanodiscs (diameter of > ca. 40 nm) can be exploited for solid‐state NMR studies on membrane proteins.     

A Usual G‐Protein‐Coupled Receptor in Unusual Membranes

G‐protein‐coupled receptors (GPCRs) are the largest family of membrane‐bound receptors and constitute about 50 % of all known drug targets. They offer great potential for membrane protein nanotechnologies. We report here a charge‐interaction‐directed reconstitution mechanism that induces spontaneous insertion of bovine rhodopsin, the eukaryotic GPCR, into both lipid‐ and polymer‐based artificial membranes. We reveal a new allosteric mode of rhodopsin activation incurred by the non‐biological membranes: the cationic membrane drives a transition from the inactive MI to the activated MII state in the absence of high [H⁺] or negative spontaneous curvature. We attribute this activation to the attractive charge interaction between the membrane surface and the deprotonated Glu134 residue of the rhodopsin‐conserved ERY sequence motif that helps break the cytoplasmic “ionic lock”. This study unveils a novel design concept of non‐biological membranes to reconstitute and harness GPCR functions in synthetic systems.     

Structural Studies of Self‐Assembled Subviral Particles: Combining Cell‐Free Expression with 110 kHz MAS NMR Spectroscopy

Viral membrane proteins are prime targets in combatting infection. Still, the determination of their structure remains a challenge, both with respect to sample preparation and the need for structural methods allowing for analysis in a native‐like lipid environment. Cell‐free protein synthesis and solid‐state NMR spectroscopy are promising approaches in this context, the former with respect to its great potential in the native expression of complex proteins, and the latter for the analysis of membrane proteins in lipids. Herein, we show that milligram amounts of the small envelope protein of the duck hepatitis B virus (DHBV) can be produced by cell‐free expression, and that the protein self‐assembles into subviral particles. Proton‐detected 2D NMR spectra recorded at a magic‐angle‐spinning frequency of 110 kHz on <500 μg protein show a number of isolated peaks with line widths comparable to those of model membrane proteins, paving the way for structural studies of this protein that is homologous to a potential drug target in HBV infection.     

Bioinspired,Size‐Tunable Self‐Assembly of Polymer–Lipid Bilayer Nanodiscs

Polymer‐based nanodiscs are valuable tools in biomedical research that can offer a detergent‐free solubilization of membrane proteins maintaining their native lipid environment. Herein, we introduce a novel ca. 1.6 kDa SMA‐based polymer with styrene:maleic acid moieties that can form nanodiscs containing a planar lipid bilayer which are useful to reconstitute membrane proteins for structural and functional studies. The physicochemical properties and the mechanism of formation of polymer‐based nanodiscs are characterized by light scattering, NMR, FT‐IR, and TEM. A remarkable feature is that nanodiscs of different sizes, from nanometer to sub‐micrometer diameter, can be produced by varying the lipid‐to‐polymer ratio. The small‐size nanodiscs (up to ca. 30 nm diameter) can be used for solution NMR spectroscopy studies whereas the magnetic‐alignment of macro‐nanodiscs (diameter of > ca. 40 nm) can be exploited for solid‐state NMR studies on membrane proteins.