Juxta‐terminal Helix Unwinding as a Stabilizing Factor to Modulate the Dynamics of Transmembrane Helices

Transmembrane helices of integral membrane proteins often are flanked by interfacial aromatic residues that can serve as anchors to aid the stabilization of a tilted transmembrane orientation. Yet, physical factors that govern the orientation or dynamic averaging of individual transmembrane helices are not well understood and have not been adequately explained. Using solid‐state ²H NMR spectroscopy to examine lipid bilayer‐incorporated model peptides of the GWALP23 (acetyl‐GGALW(LA)₆LWLAGA‐amide) family, we observed substantial unwinding at the terminals of several tilted helices spanning the membranes of DLPC, DMPC, or DOPC lipid bilayers. The fraying of helix ends might be vital for defining the dynamics and orientations of transmembrane helices in lipid bilayer membranes.     

Exploring the Ability of Cyclic Peptides to Target SAM Domains: A Computational and Experimental Study

Sterile alpha motif (SAM) domains are protein interaction modules with a helical fold. SAM–SAM interactions often adopt the mid-loop (ML)/end-helix (EH) model, in which the C-terminal helix and adjacent loops of one SAM unit (EH site) bind the central regions of another SAM domain (ML site). Herein, an original strategy to attack SAM–SAM associations is reported. It relies on the design of cyclic peptides that target a region of the SAM domain positioned at the bottom side of the EH interface, which is thought to be important for the formation of a SAM–SAM complex. This strategy has been preliminarily tested by using a model system of heterotypic SAM–SAM interactions involving the erythropoietin-producing hepatoma kinase A2 (EphA2) receptor and implementing a multidisciplinary plan made up of computational docking studies, experimental interaction assays (by NMR spectroscopy and surface plasmon resonance techniques) and conformational analysis (by NMR spectroscopy and circular dichroism). This work further highlights how only a specific balance between flexibility and rigidity may be needed to generate modulators of SAM–SAM interactions.     

The Molecular Bases of the Interaction between a Saponin from the Roots of Gypsophila paniculata L. and Model Lipid Membranes

In view of the possible medical applications of saponins, the molecular structure of a GOTCAB saponin from the roots of Gypsophila paniculata L. was determined by NMR. The biological activity of saponins may depend on the interaction with cell membranes. To obtain more insight in the mechanism of membrane-related saponin function, an experimental and theoretical study was conducted. Ternary lipid systems composed of sphingomyelin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and cholesterol were used as models of mammalian cell membranes. The membrane–saponin interaction was studied experimentally by monitoring surface pressure in the monomolecular films formed at the air–aqueous subphase interface. The behavior of GOTCAB saponin in a water box and model monolayer systems was characterized by molecular dynamics simulations. The results obtained showed that, in the systems used, cholesterol had a decisive effect on the interaction between GOTCAB and phosphocholine or sphingomyelin as well as on its location within the lipid film.     

Solution‐NMR Characterization of Outer‐Membrane Protein A from E. coli in Lipid Bilayer Nanodiscs and Detergent Micelles

X‐ray crystallography and solution NMR of detergent‐reconstituted OmpA (outer membrane protein A from E. coli) had shown that this protein forms an eight‐stranded transmembrane β‐barrel, but only limited information was obtained for the extracellular loops. In NMR studies of OmpA in two different detergent micelles, “NMR‐invisible” amino acid residues in‐between the extracellular loops and the β‐barrel prevented complete structural characterization. Here, we show that this NMR‐invisible ring around the β‐barrel of OmpA is also present in lipid bilayer nanodiscs and in mixed micelles with a third detergent, thus suggesting that the implicated rate processes have a functional role rather than representing an artifact of the protein reconstitution. In addition to sequence‐specific NMR assignments for OmpA in the nanodiscs, the present results are based on a protocol of micro‐coil TROSY‐ and CRINEPT‐type NMR diffusion measurements for studying the hydrodynamic properties and the foldedness of [²H,¹⁵N]‐labeled membrane proteins in nanodiscs. This protocol can be applied under conditions closely similar to those used for NMR structure determinations or crystallization trials.     

Effect of Dietary Cholesterol and Cholesterol Oxides on Blood Cholesterol,Lipids, and the Development of Atherosclerosis in Rabbits

Two studies were conducted to determine the effects of dietary cholesterol (CHO) and cholesterol oxides (COPs) on the development of atherosclerosis and the changes in fatty acid and blood characteristics in rabbits. In the first study, forty male New Zealand white rabbits were divided into 5 groups and fed commercial rabbit chow with no added CHO or COPs, 1 g CHO, 0.9 g CHO + 0.1 g COPs, 0.8 g CHO + 0.2 g COPs, or 0.5 g CHO + 0.5 g COPs per kg diet. In the second study, 24 male New Zealand White rabbits were divided into 3 groups and fed a diet containing 2 g CHO, 1.6 g CHO + 0.4 g COPs, or 1.2 g CHO + 0.8 g COPs per kg diet. All diets induced atherosclerotic lesions in the rabbits’ ascending thoracic aorta. The serum CHO and triglyceride levels (p < 0.05) increased significantly with the increased levels of CHO in the diets. Dietary CHO or COPs did not influence high-density lipoprotein CHO levels. The ratio of saturated fatty acid to unsaturated fatty acid increased as the level of dietary CHO and COPs increased.     

Fast NMR Methods for Measuring in the Direct and/or Competition Mode the Dissociation Constants of Chemical Fragments Interacting with a Receptor

Ligand-based NMR screening represents a powerful method in fragment-based drug discovery for the identification of chemical matter interacting with the receptor of interest. The large dynamic range of these methods allows the detection of weakly binding ligands. However, the methodology has not been extensively used for quantifying the strength of these interactions. This knowledge is important for ranking fragments according to their binding strength and for prioritizing structure-based and medicinal chemistry activities. Rapid NMR methods for measuring the dissociation constant in direct and competition modes are presented here. The theory underpinning these methods are presented, along with their application to the measurement of the binding affinities of several ligands of the heat shock protein 90.     

Role of STAR and SCP2/SCPx in the Transport of Cholesterol and Other Lipids

Cholesterol is a lipid molecule essential for several key cellular processes including steroidogenesis. As such, the trafficking and distribution of cholesterol is tightly regulated by various pathways that include vesicular and non-vesicular mechanisms. One non-vesicular mechanism is the binding of cholesterol to cholesterol transport proteins, which facilitate the movement of cholesterol between cellular membranes. Classic examples of cholesterol transport proteins are the steroidogenic acute regulatory protein (STAR; STARD1), which facilitates cholesterol transport for acute steroidogenesis in mitochondria, and sterol carrier protein 2/sterol carrier protein-x (SCP2/SCPx), which are non-specific lipid transfer proteins involved in the transport and metabolism of many lipids including cholesterol between several cellular compartments. This review discusses the roles of STAR and SCP2/SCPx in cholesterol transport as model cholesterol transport proteins, as well as more recent findings that support the role of these proteins in the transport and/or metabolism of other lipids.     

NMR chemical shift perturbation study of the N-terminal domain of Hsp90 upon binding of ADP,AMP-PNP,geldanamycin, and radicicol

Hsp90 is one of the most abundant chaperone proteins in the cytosol. In an ATP-dependent manner it plays an essential role in the folding and activation of a range of client proteins involved in signal transduction and cell cycle regulation. We used NMR shift perturbation experiments to obtain information on the structural implications of the binding of AMP-PNP (adenylyl-imidodiphosphate-a non-hydrolysable ATP analogue), ADP and the inhibitors radicicol and geldanamycin. Analysis of (1)H,(15)N correlation spectra showed a specific pattern of chemical shift perturbations at N210 (ATP binding domain of Hsp90, residues 1-210) upon ligand binding. This can be interpreted qualitatively either as a consequence of direct ligand interactions or of ligand-induced conformational changes within the protein. All ligands show specific interactions in the binding site, which is known from the crystal structure of the N-terminal domain of Hsp90. For AMP-PNP and ADP, additional shift perturbations of residues outside the binding pocket were observed and can be regarded as a result of conformational rearrangement upon binding. According to the crystal structures, these regions are the first alpha-helix and the "ATP-lid" ranging from amino acids 85 to 110. The N-terminal domain is therefore not a passive nucleotide-binding site, as suggested by X-ray crystallography, but responds to the binding of ATP in a dynamic way with specific structural changes required for the progression of the ATPase cycle.     

Influence of Alkyl Chain Length on Thermal Properties,Structure, and Self-Diffusion Coefficients of Alkyltriethylammonium-Based Ionic Liquids

The application of ionic liquids (ILs) has grown enormously, from their use as simple solvents, catalysts, media in separation science, or electrolytes to that as task-specific, tunable molecular machines with appropriate properties. A thorough understanding of these properties and structure–property relationships is needed to fully exploit their potential, open new directions in IL-based research and, finally, properly implement the appropriate applications. In this work, we investigated the structure–properties relationships of a series of alkyltriethylammonium bis(trifluoromethanesulfonyl)imide [TEA-R][TFSI] ionic liquids in relation to their thermal behavior, structure organization, and self-diffusion coefficients in the bulk state using DSC, FT-IR, SAXS, and NMR diffusometry techniques. The phase transition temperatures were determined, indicating alkyl chain dependency. Fourier-transformed infrared spectroscopy studies revealed the structuration of the ionic liquids along with alkyl chain elongation. SAXS experiments clearly demonstrated the existence of polar/non-polar domains. The alkyl chain length influenced the expansion of the non-polar domains, leading to the expansion between cation heads in polar regions of the structured IL. ¹H NMR self-diffusion coefficients indicated that alkyl chain elongation generally caused the lowering of the self-diffusion coefficients. Moreover, we show that the diffusion of anions and cations of ILs is similar, even though they vary in their size.     

Effect of Dietary Cholesterol and Fat on Cell Cholesterol Transfer to Postprandial Plasma in Hyperlipidemic Men

Postprandial chylomicrons are potent ultimate acceptors of cell membrane cholesterol and are believed to accelerate reverse cholesterol transport (RCT). We compared the effects of meals rich in polyunsaturated fat (PUFA) and either high (605 mg) or low (151 mg) in cholesterol and a meal rich in dairy fat (DF) in the form of cream on net in vitro transport of red blood cell (RBC) membrane cholesterol to 4 and 6 h postprandial plasma in eight normotriglyceridemic (NTG-H) and eight hypertriglyceridemic (HTG-H) men with mild to moderate hypercholesterolemia. In HTG-H men, cell cholesterol accumulation in 6-h postprandial plasma was significantly (P = 0.02) less after the PUFA-HC meal compared with the other meals. The significant (P < 0.001) increase in cell plus endogenous cholesterol accumulation in the triglyceride-rich lipoprotein (TRL) fraction of 4 h postprandial plasma incubated with RBC was significantly (P = 0.007) higher after the PUFA-HC meal compared with DF meal in HTG-H men. In NTG-H men, cholesterol accumulation in plasma and plasma lipoproteins in the presence and absence of RBC was not significantly affected by the type of meal ingested. These data suggest that addition of large amounts of cholesterol to a PUFA meal may impair diffusion-mediated transport of cell membrane cholesterol to postprandial plasma and that replacing DF with PUFA in a meal increases postprandial lipemia and may potentially increase cholesterol accumulation in atherogenic postprandial TRL in HTG-H men.     

Diversity of Lipid Function in Atherogenesis: A Focus on Endothelial Mechanobiology

Atherosclerosis is one of the most important problems in modern medicine. Its high prevalence and social significance determine the need for a better understanding of the mechanisms of the disease’s development and progression. Lipid metabolism and its disorders are one of the key links in the pathogenesis of atherosclerosis. Lipids are involved in many processes, including those related to the mechanoreception of endothelial cells. The multifaceted role of lipids in endothelial mechanobiology and mechanisms of atherogenesis are discussed in this review. Endothelium is involved in ensuring adequate vascular hemodynamics, and changes in blood flow characteristics are detected by endothelial cells and affect their structure and function.     

The Effect of the Osmotically Active Compound Concentration Difference on the Passive Water and Proton Fluxes across a Lipid Bilayer

The molecular details of the passive water flux across the hydrophobic membrane interior are still a matter of debate. One of the postulated mechanisms is the spontaneous, water-filled pore opening, which facilitates the hydrophilic connection between aqueous phases separated by the membrane. In the paper, we provide experimental evidence showing that the spontaneous lipid pore formation correlates with the membrane mechanics; hence, it depends on the composition of the lipid bilayer and the concentration of the osmotically active compound. Using liposomes as an experimental membrane model, osmotically induced water efflux was measured with the stopped-flow technique. Shapes of kinetic curves obtained at low osmotic pressure differences are interpreted in terms of two events: the lipid pore opening and water flow across the aqueous channel. The biological significance of the dependence of the lipid pore formation on the concentration difference of an osmotically active compound was illustrated by the demonstration that osmotically driven water flow can be accompanied by the dissipation of the pH gradient. The application of the Helfrich model to describe the probability of lipid pore opening was validated by demonstrating that the probability of pore opening correlates with the membrane bending rigidity. The correlation was determined by experimentally derived bending rigidity coefficients and probabilities of lipid pores opening.     

Recognition of planar and nonplanar ligands in the malachite green-RNA aptamer complex

Ribonucleic acids are an attractive drug target owing to their central role in many pathological processes. Notwithstanding this potential, RNA has only rarely been successfully targeted with novel drugs. The difficulty of targeting RNA is at least in part due to the unusual mode of binding found in most small-molecule-RNA complexes: the ligand binding pocket of the RNA is largely unstructured in the absence of ligand and forms a defined structure only with the ligand acting as scaffold for folding. Moreover, electrostatic interactions between RNA and ligand can also induce significant changes in the ligand structure due to the polyanionic nature of the RNA. Aptamers are ideal model systems to study these kinds of interactions owing to their small size and the ease with which they can be evolved to recognize a large variety of different ligands. Here we present the solution structure of an RNA aptamer that binds triphenyl dyes in complex with malachite green and compare it with a previously determined crystal structure of a complex formed with tetramethylrosamine. The structures illustrate how the same RNA binding pocket can adapt to accommodate both planar and nonplanar ligands. Binding studies with single- and double-substitution mutant aptamers are used to correlate three-dimensional structure with complex stability. The two RNA-ligand complex structures allow a discussion of structural changes that have been observed in the ligand in the context of the overall complex structure. Base pairing and stacking interactions within the RNA fold the phosphate backbone into a structure that results in an asymmetric charge distribution within the binding pocket that forces the ligand to adapt through a redistribution of the positive partial charge.     

Conformation, dynamics, and insertion of a noncysteine-containing protegrin-1 analogue in lipid membranes from solid-state NMR spectroscopy

Disulfide-bonded beta-hairpin structures are common among antimicrobial peptides. Disulfide bonds are known to be important for antimicrobial activity, but the underlying structural reason is not well understood. We have investigated the membrane-bound structure of a disulfide-deleted analogue of the antimicrobial peptide protegrin-1, in which the four Cys residues were replaced by Ala. The secondary structure, dynamics, and topology of this Ala-PG1 peptide in the membrane were determined by using magic-angle-spinning NMR spectroscopy. Conformation-dependent (13)C isotropic chemical shifts of multiple (13)C-labeled residues were obtained from 1D cross-polarization and direct-polarization spectra, and from 2D J-coupling-mediated (13)C-(13)C correlation spectra. Most labeled residues exhibited two conformations: a random coil and a beta-sheet structure. The dual-conformation property was present in both anionic lipid bilayers, which mimic the bacterial membrane, and zwitterionic cholesterol-containing bilayers, which mimic the eukaryotic cell membrane. The mobility of the peptide was measured by using a 2D C-H dipolar-shift correlation experiment. The random-coil fraction was highly mobile whereas the beta-sheet component was rigid. (1)H spin diffusion from the lipid chains to the peptide indicates that the beta-sheet component was well inserted into the anionic membrane, but surface bound in the cholesterol-containing neutral membrane. Thus, the removal of disulfide bonds changed some PG-1 molecules to highly mobile random coils that were poorly associated with the lipid membrane, but other molecules retained a beta-sheet conformation and had a similar membrane-binding topology to the parent peptide. Thus, the reduced antimicrobial activity of Ala-PG1 was largely due to the reduced number of insertion-competent beta-sheet molecules, rather than uniformly weakened activity of identically structured peptides.     

Effect of Amphipathic HIV Fusion Inhibitor Peptides on POPC and POPC/Cholesterol Membrane Properties: A Molecular Simulation Study

T-20 and T-1249 fusion inhibitor peptides were shown to interact with 1-palmitoyl-2-oleyl-phosphatidylcholine (POPC) (liquid disordered, ld) and POPC/cholesterol (1:1) (POPC/Chol) (liquid ordered, lo) bilayers, and they do so to different extents. Although they both possess a tryptophan-rich domain (TRD), T-20 lacks a pocket binding domain (PBD), which is present in T-1249. It has been postulated that the PBD domain enhances FI interaction with HIV gp41 protein and with model membranes. Interaction of these fusion inhibitor peptides with both the cell membrane and the viral envelope membrane is important for function, i.e., inhibition of the fusion process. We address this problem with a molecular dynamics approach focusing on lipid properties, trying to ascertain the consequences and the differences in the interaction of T-20 and T-1249 with ld and lo model membranes. T-20 and T-1249 interactions with model membranes are shown to have measurable and different effects on bilayer structural and dynamical parameters. T-1249’s adsorption to the membrane surface has generally a stronger influence in the measured parameters. The presence of both binding domains in T-1249 appears to be paramount to its stronger interaction, and is shown to have a definite importance in membrane properties upon peptide adsorption.     

Structural Insight into the Complex of Ferredoxin and [FeFe] Hydrogenase from Chlamydomonas reinhardtii

The transfer of photosynthetic electrons by the ferredoxin PetF to the [FeFe] hydrogenase HydA1 in the microalga Chlamydomonas reinhardtii is a key step in hydrogen production. Electron delivery requires a specific interaction between PetF and HydA1. However, because of the transient nature of the electron‐transfer complex, a crystal structure remains elusive. Therefore, we performed protein–protein docking based on new experimental data from a solution NMR spectroscopy investigation of native and gallium‐substituted PetF. This provides valuable information about residues crucial for complex formation and electron transfer. The derived complex model might help to pinpoint residue substitution targets for improved hydrogen production.     

Effect of Ecdysterone on the Hepatic Transcriptome and Lipid Metabolism in Lean and Obese Zucker Rats

Conflicting reports exist with regard to the effect of ecdysterone, the predominating representative of steroid hormones in insects and plants, on hepatic and plasma lipid concentrations in different rodent models of obesity, fatty liver, and diabetes, indicating that the effect is dependent on the rodent model used. Here, the hypothesis was tested for the first time that ecdysterone causes lipid-lowering effects in genetically obese Zucker rats. To test this hypothesis, two groups of male obese Zucker rats (n = 8) were fed a nutrient-adequate diet supplemented without or with 0.5 g ecdysterone per kg diet. To study further if ecdysterone is capable of alleviating the strong lipid-synthetic activity in the liver of obese Zucker rats, the study included also two groups of male lean Zucker rats (n = 8) which also received either the ecdysterone-supplemented or the non-supplemented diet. While hepatic and plasma concentrations of triglycerides and cholesterol were markedly higher in the obese compared to the lean rats (p < 0.05), hepatic and plasma triglyceride and cholesterol concentrations did not differ between rats of the same genotype fed the diets without or with ecdysterone. In conclusion, the present study clearly shows that ecdysterone supplementation does not exhibit lipid-lowering actions in the liver and plasma of lean and obese Zucker rats.