Lipid Bound Research Articles

HPLC-QQQ-MS/MS-based authentication and determination of free and bound sialic acids content in human, bovine, sheep, goat milk, and infant formula.

An accurate method for qualitative and quantitative analysis of lipid-bound (LB), protein-bound (PB), oligosaccharides-bound, and free sialic acids in milk was developed by using high-performance liquid chromatography -triple quadrupole-tandem mass spectrometer. The profile of free and bound sialic acids in milk (human, bovine, goat, and sheep) and infant formula (IF) was examined in the present study. Human milk contains only N-acetylneuraminic acid (Neu5Ac) and was mainly present in the form of oligosaccharide-bound. The content of total Neu5Ac (T-Neu5Ac), free and bound Neu5Ac in human milk decreased with the prolongation of lactation. The most intriguing finding was the increase in the proportion of PB and LB sialic acids. The sialic acids in bovine and sheep milk were mainly PB and oligosaccharides-bound Neu5Ac. T-Neu5Ac in goat milk (GM) was 67.44-89.72µg/mL and was mainly PB Neu5Ac, but total N-glycolylneuraminic acid (T-Neu5Gc) content of GM can be as high as 100.01µg/mL. The concentration of T-Neu5Gc in sheep and GM was significantly higher than that of bovine milk (BM). T-Neu5Gc content of GM -based IF was 264.86µg/g, whereas T-Neu5Gc content of BM -based IF was less (2.26-17.01µg/g). Additionally, our results found that there were also sialic acids in IF ingredients, which were mainly bound with protein and oligosaccharides, primarily derived from desalted whey powder and whey protein concentrate.

Native mass spectrometry reveals the simultaneous binding of lipids and zinc to rhodopsin

Rhodopsin, a prototypical G-protein-coupled receptor, is responsible for scoptic vision at low-light levels. Although rhodopsin’s photoactivation cascade is well understood, it remains unclear how lipid and zinc binding to the receptor are coupled. Using native mass spectrometry, we developed a novel data analysis strategy to deconvolve zinc and lipid bound to the proteoforms of rhodopsin and investigated the allosteric interaction between lipids and zinc binding. We discovered that phosphatidylcholine bound to rhodopsin with a greater affinity than phosphatidylserine or phosphatidylethanolamine, and that binding of all lipids was influenced by zinc but with different effects. In contrast, zinc binding was relatively unperturbed by lipids. Overall, these data reveal that lipid binding can be strongly and differentially influenced by metal ions.

Structural insights into the lipid transfer mechanism of a non-specific lipid transfer protein.

The non-specific lipid transfer proteins (nsLTPs) are multifunctional seed proteins engaged in several different physiological processes. The nsLTPs are stabilized by four disulfide bonds and exhibit a characteristic hydrophobic cavity, which is the primary lipid binding site. While these proteins are known to transfer lipids between membranes, the mechanism of lipid transfer has remained elusive. Four crystal structures of nsLTP from Solanum melongena, one in the apo-state and three myristic acid bound states were determined. Among the three lipid bound states, two lipid molecules were bound on the nsLTP surface at different positions and one was inside the cavity. The lipid-dependent conformational changes leading to opening of the cavity were revealed based on structural and spectroscopic data. The surface-bound lipid represented a transient intermediate state and the lipid ultimately moved inside the cavity through the cavity gate as revealed by molecular dynamics simulations. Two critical residues in the loop regions played possible 'gating' role in the opening and closing of the cavity. Antifungal activity and membrane permeabilization effect of nsLTP against Fusarium oxysporum suggested that it could possibly involve in bleaching out the lipids. Collectively, these studies support a model of lipid transfer mechanism by nsLTP via intermediate states.

A Soluble Fluorescent Binding Assay Reveals PIP2 Antagonism of TREK-1 Channels

SUMMARYLipid regulation of ion channels by low-abundance signaling lipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) has emerged as a central cellular mechanism for controlling ion channels and the excitability of nerves. A lack of robust assays suitable for facile detection of a lipid bound to a channel has hampered the probing of the lipid binding sites and measuring the pharmacology of putative lipid agonists for ion channels. Here, we show a fluorescent PIP2 competition assay for detergent-purified potassium channels, including TWIK-1-related K+-channel (TREK-1). Anionic lipids PA and phosphatidylglycerol (PG) bind dose dependently (9.1 and 96 mM, respectively) and agonize the channel. Our assay shows PIP2 binds with high affinity (0.87 mM) but surprisingly can directly antagonize TREK-1 in liposomes. We propose a model for TREK-1 lipid regulation where PIP2 can compete with PA and PG agonism based on the affinity of the lipid for a site within the channel.

Abstract 223: Optimization of Apolipoprotein A-1 Mimetic Peptides for Lipid Binding and Lecithin-cholesterol Acyltransferase Activation

Introduction: Apolipoprotein A-I (apoA-I) mimetic peptides present an exciting avenue in the clinical development of synthetic HDL (sHDL), offering a more feasible and economical approach in sHDL production. One such example is ETC-642, or 22A—a 22 amino acid ApoA-I mimetic peptide tested in single and multiple dose clinical trials. The goal of this study is to understand how specific amino acid substitutions affect physico-chemical properties of 22-mer peptides and correlate them with the ability to form stable nano-sized sHDL particles and promote LCAT activity. Methods/Results: For this study, we used the 22A peptide (PVLDLFNELLNELLEALKQKLK or ETC-642) and designed 10 variants comprised of a single amino acid substitution, denoted 22A-1-10. We used computational programs FMAP (Folding of Membrane-Associated Peptides), PPM2.0 and FRAMEWORK to select peptides that are predicted to form α-helices, have good membrane penetration (>7.8 Å) with low transfer energy to membranes (ΔG transfer <-16.1 kcal/mol), and form stable α-helices (ΔG helix <-23.5 kcal/mol) with strong intermolecular LCAT interactions. The calculated ΔG helix varied from -18.4 to -24.4 kcal/mol, and G transfer varied from -11.1 to -17.2 kcal/mol for the synthesized peptides. The percent peptide α-helicity in lipid bound form was measured by circular dichroism spectroscopy and was found to vary dramatically between 19 and 82%. The lipid solubilization kinetics were determined using UV absorbance to measure turbidity. The particle size and stability of resulting HDL particles were measured by dynamic light scattering and gel permeation chromatography. The peptides with low predicted energy of lipid binding exhibited high helicity and formed stable HDL particles with sizes less than 20 nm. The HDLs formed from apoA-I peptides with high helicity were also able to activate LCAT and facilitate sterol esterification with a K cat of 5 to 30 min -1 . Conclusions: Our data shows that that minimalistic changes in primary structure of ApoA-1 peptide can lead significant changes in physico-chemical properties. Peptides with degree of α-helicity exhibited superior lipid binding and formed stable HDL particles of small size able to interact with LCAT.

Membrane proteins bind lipids selectively to modulate their structure and function.

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments1-7 and that lipids can bind to specific sites, for example in potassium channels8. Fundamental questions remain however regarding the extent of membrane protein selectivity toward lipids. Here we report a mass spectrometry (MS) approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL), aquaporin Z (AqpZ), and the ammonia channel (AmtB) using ion mobility MS (IM-MS), which reports gas-phase collision cross sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas-phase. By resolving lipid-bound states we then rank bound lipids based on their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Results show that lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability, the highest-ranking lipid however is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation9. AqpZ is also stabilized by many lipids with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays, we discover that cardiolipin modulates AqpZ function. Analogous experiments identify AmtB as being highly selective for phosphatidylglycerol prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that reposition AmtB residues to interact with the lipid bilayer. Overall our results demonstrate that resistance to unfolding correlates with specific lipid-binding events enabling distinction of lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be influential not only for defining the selectivity of membrane proteins toward lipids but also for understanding the role of lipids in modulating function or drug binding.

Binding of Cationic Lipids to Milk β-Lactoglobulin

We determined the bindings of several lipids such as cholesterol (CHOL), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethyl-ammoniumbromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE) to β-lactoglobulin (β-LG) at physiological conditions. FTIR, CD, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of lipid-protein complexes. Structural analysis showed that lipids bind β-LG via both hydrophilic and hydrophobic interactions with overall binding constants of K(CHOL-β-LG) = 6.0 (±0.6) × 10(3) M(-1), K(DOPE-β-LG) = 6.5 (±0.7) × 10(3) M(-1), K(DDAB-β-LG) = 1.6 (±0.3) × 10(4) M(-1), and K(DOTAP-β-LG) = 2.2 (±0.67) × 10(4) M(-1). The number of lipid bound per protein molecule (n) was 0.8 (CHOL), 0.7 (DOPE), 1.0 (DDAB), and 1.3 (DOTAP). Molecular modeling showed the participation of several amino acid residues in lipid-protein complexation with the order of binding DOTAP > DDAB > DOPE > CHOL. Alterations of the protein conformation were observed in the presence of lipids with a minor decrease in β-sheet and an increase in turn structure.

Structural and Functional Insights Into Lipid Binding by Oligomeric Alpha-Synuclein

Oligomeric alpha-synuclein is considered to be the potential toxic species responsible for the onset and progression of Parkinson's disease, possibly through the disruption of lipid membranes. Although there is evidence that oligomers contain considerable amounts of secondary structure, more detailed information on the structural characteristics and how these mediate oligomer-lipid binding are critically lacking. We have used tryptophan fluorescence spectroscopy to gain insight into the structural features of oligomeric alpha-synuclein and the structural basis for oligomer-lipid interactions. Several single tryptophan mutants of alpha-synuclein were used to gain site-specific information about the microenvironment of monomeric, oligomeric and lipid bound oligomeric alpha-synuclein. Acrylamide quenching and spectral analyses indicate that the tryptophan residues are considerably more solvent protected in the oligomeric form compared to the monomeric protein. In the oligomers, the negatively charged C-terminus was the most solvent exposed part of the protein. Upon lipid binding a blue shift in fluorescence is observed for alpha-synuclein mutants where the tryptophan is located within the N-terminal region. These results suggest that as in the case of monomeric alpha-synuclein, the N-terminus is critical in determining oligomer-lipid binding. We have further systematically studied the influence of the physical membrane properties and solution conditions on lipid bilayer disruption by oligomeric alpha-synuclein using a dye release assay, and have quantitatively studied oligomer lipid binding by confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS). The results indicate that the oligomeric species specifically bind to negatively charged lipids in the liquid disordered phase.

Apolipophorin III lysine modification: Effect on structure and lipid binding

Apolipophorin III (apoLp-III) from Locusta migratoria was used as a model to investigate apolipoprotein lipid binding interactions. ApoLp-III contains eight lysine residues, of which seven are located on one side of the protein. To investigate the role of positive charges on lipid binding, lysine residues were acetylated by acetic anhydride. The degree of acetylation was analyzed by SDS-PAGE and MALDI-TOF, indicating a maximum of eight acetyl additions. Modified apoLp-III remained α-helical, but displayed a decreased α-helical content (from 78 to 54%). Acetylation resulted in a slight increase in protein stability, as indicated by a change in the midpoint of guanidine–HCl induced denaturation from 0.55 (unmodified) to 0.65 M (acetylated apoLp-III). Lipid bound apoLp-III, either acetylated or unmodified, displayed similar increases in helical content and midpoint of guanidine–HCl-induced denaturation of ∼ 4 M. The ability to solubilize vesicles of dimyristoylphosphatidylcholine remained unchanged. However, the rate to solubilize dimyristoylphosphatidylglycerol vesicles was reduced two-fold. In addition, a decreased ability to stabilize diacylglycerol-enriched low density lipoproteins was observed. This indicated that lysine residues are not critical for the protein's ability to bind to zwitterionic phospholipids. Since binding interactions with ionic phospholipids and lipoproteins were affected by acetylation, lysine side-chains may play a modulating role in the interaction with more complex lipid surfaces encountered in vivo.

Voltammetric and spectroscopic studies on methyl green and cationic lipid bound to calf thymus DNA

DNA interaction with cationic lipids promises to be a versatile and effective synthetic transfection agent. This paper presents the study on binding of a simple artifical cationic lipid, cetyltrimethylammonium bromide (CTAB), to calf thymus DNA (CT DNA) prior to the condensation process, taking methyl green (MG) as a probe. The results show that the CTAB binds to DNA through electrostatic interaction forming a hydrophobic complex, thus changing the micro-environment of duplex of DNA, so the binding state of MG and DNA is changed, and a complex CTAB-CT DNA-MG is formed. This fact suggests a new way to mediate the conformation of molecular assemblies of DNA and lipids.

Role of charged amino acid side chains in the stability and lipid binding of Manduca sexta apolipophorin III

AbstractChemical modification procedures were employed to neutralize charged amino acid side chains of apolipophorin III (apoLp‐III). Glutamate plus aspartate carboxylate side chains were amidated while, in other experiments, the ϵ‐amino groups of lysine residue side chains were acetylated. Circular dichroism (CD) spectroscopy was performed to assess the effect of chemical modification on the secondary structure of apoLp‐III. Compared to control, unmodified apoLp‐III, both amidated and acetylated apoLp‐IIIs possessed significantly diminished levels of α‐helical structure. A similarly significant amount of α‐helix structure could be induced in both modified apoLp‐IIIs, however, by the addition of 50% trifluoroethanol, a helix inducing solvent, indicating that the proteins have retained their capacity to form helical secondary structures. The lipid binding interactions of chemically modified apoLp‐IIIs were also examined in lipoprotein binding assays. Whereas control, unmodified apoLp‐III displayed lipid binding activity, neither modified apoLp‐III was capable of interaction with the substrate lipid surface. In phospholipid binding assays using the model compound, dimyristoylphosphatidylcholine, acetylated apoLp‐III failed to interact while amidated apoLp‐III showed limited interaction. When sodium dodecyl sulfate (SDS) micelles were employed as a model lipid surface, interaction of the modified apoLp‐IIIs was observed. To characterize the relative stability of the interaction of control and modified apoLp‐IIIs with SDS micelles, urea denaturation studies were performed. These experiments showed that, while control and amidated apoLp‐IIIs were relatively resistant to urea induced denaturation, acetylated apoLp‐III was susceptible. Taken as a whole, the results suggest that charged amino acid residues play an important role in stabilization of the lipid‐free helix bundle conformation of apoLp‐III and may promote stabilization of the lipid bound state through charge‐charge interactions with lipoprotein surface components. © 1995 Wiley‐Liss, Inc.

Lipid-dependent membrane enzymes. A kinetic model for cooperative activation in the absence of cooperativity in lipid binding.

The dependence of integral membrane enzymes on lipid activators in analyzed in terms of multiple binding site kinetics. Rate equations for an enzyme with n independent and indentical lipid binding sites are derived for the case that enzyme activity is proportional to the total amount of lipid bound, or that only fully substituted enzyme is active. A third equation applies to the case that lipids bind with infinite cooperativity to give fully substituted and active enzyme. None of the three models was entirely consistent with existing experimental data. The following kinetic model is shown to accommodate the degree of cooperativity observed in lipid activation experiments as well as the number of independent lipid-binding sites determined by electron-spin resonance measurements. The membrane enzyme is assumed to have n non-interacting and identical lipid-binding sites. Only fully substituted enzyme (ELn) and the next most highly substituted forms such as ELn-1 and ELn-2 may possess enzyme activity. These assumptions lead to cooperativity in activation. Cooperativity reaches a maximum when enzyme activity starts to appear with about 80% of the full lipid substitution. The increase in cooperativity is accompanied by a decrease in the lipid concentration required for half-maximal activation. Further kinetic aspects of a dynamic boundary lipid layer around integral membrane enzymes are discussed.

Binding and extractability of wheat flour lipid after dough formation.

AbstractObservations that lipids normally extracted from flour with nonpolar solvents become bound upon dough or gluten formation have been confirmed. Lipids bound in this way can be extracted by solvents of high dielectric constant and by mixtures of some alcohols with solvents containing donor hydrogen atoms. Low molar volume of solvent, in general associated with high dielectric constant, also leads to good extraction of bound lipid. Ethanol/ether/water (2:2:1) was the most efficient of the solvents tried and appeared to give almost complete extraction of lipid from flour.The reported reduction of lipid bound in dough containing salt has been investigated further and it was found that lipid binding decreased with added salt up to about 4% of flour weight. Doughing and lipid‐binding occur when flour is mixed with solvents of high dielectric constant as well as with water.