Magnetic resonance investigations of lipid motion in isotropic bicelles

The dynamics of DMPC in different isotropic bicelles have been investigated by NMR and EPR methods. The local dynamics were obtained by interpretation of 13C NMR relaxation measurements of DMPC in the bicelles, and these results were compared to EPR spectra of spin-labeled lipids. The overall size of the bicelles was investigated by PFG NMR translational diffusion measurements. The dynamics and relative sizes were compared among three different bicelles: [DMPC]/[DHPC] = 0.25, [DMPC]/[DHPC] = 0.5, and [DMPC]/[CHAPS] = 0.5. The local motion is found to depend much more strongly on the choice of the detergent, rather than the overall size of the bicelle. The results provide an explanation for differences in apparent dynamics for different peptides, which are bound to bicelles. This in turn determines under what conditions reasonable NMR spectra can be observed. A model is presented in which extensive local motion, in conjunction with the overall size, affects the spectral properties. An analytical expression for the size dependence of the bicelles, relating the radius of the bilayer region with physical properties of the detergent and the lipid, is also presented.     

Urea Unfolding of Opsin in Phospholipid Bicelles†

Opsin is the unstable apo‐protein of the light‐activated G protein‐coupled receptor rhodopsin. We investigated the stability of bovine opsin, solubilized in 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC)/detergent bicelles, against urea‐induced unfolding. A single irreversible protein unfolding transition was observed from changes in intrinsic tryptophan fluorescence and far‐UV circular dichroism. This unfolding transition correlated with loss of protein activity. Changes in tertiary structure, as indicated by fluorescence measurements, were concomitant with an approximate 50% reduction in α‐helical content of opsin, indicating that global unfolding had been induced by urea. The urea concentration at the midpoint of unfolding was dependent on the lipid/detergent environment, occurring at approximately 1.2 m urea in DMPC/1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine bicelles, while being significantly stabilized to approximately 3.5 m urea in DMPC/3‐[(cholamidopropyl)dimethylammonio]‐1‐propanesulfonate bicelles. These findings demonstrate that interactions with the surrounding lipids and detergent are highly influential in the unfolding of membrane protein structure. The urea/bicelle system offers the possibility for a more detailed understanding of the structural changes that take place upon irreversible unfolding of opsin.     

Unprecedented observation of days-long remnant orientation of phospholipid bicelles: a small-angle X-ray scattering and theoretical study

Nanometric bilayer-based self-assembled micelles commonly named as bicelles, formed with a mixture of long and short chains phosphatidylcholine lipids (PC), are known to orient spontaneously in a magnetic field. This field-induced orientational order strongly depends on the molecular structure of the phospholipids. Using small-angle X-ray scattering (SAXS), we performed detailed structural studies of bicelles and investigated the orientation/relaxation kinetics in three different systems: saturated-chain lipid bicelles made of DMPC (dimyristoyl PC)/DCPC (1,2-dicaproyl PC) with and without the added paramagnetic lanthanide ions Eu(3+), as well as bicelles of TBBPC (1-tetradecanoyl-2-(4-(4-biphenyl)butanoyl)-sn-glycero-3-PC)/DCPC. The structural study confirmed the previous NMR studies, which showed that DMPC bicelles orient with the membrane normal perpendicular (defined here as "nematic" orientation) to the magnetic field, whereas they orient parallel (defined here as "smectic" orientation) to the magnetic field in the presence of Eu(3+). The TBBPC bicelles also show smectic orientation. Surprisingly, the orientational order induced in the magnetic field remains even after the magnetic field is removed, which allowed us to investigate the orientation and relaxation kinetics of different bicelle structures. We demonstrate that this kinetics is very different for all three types of bicelles at the same lipid concentration; DMPC bicelles (~40 nm diameter) with and without Eu(3+) orient faster than TBBPC bicelles (~80 nm diameter). However, for the relaxation, DMPC bicelles (nematic) lose their macroscopic orientation only after one hour, whereas both DMPC bicelles with Eu(3+) and TBBPC bicelles (smectic) remarkably stay oriented for up to several days! These results indicate that the orientation mechanism of these nanometric disks in the magnetic field is governed by their size, with smaller bicelles orienting faster than the larger bicelles. Their relaxation mechanism outside the magnetic field, however, is governed by the degree of ordering. Indeed, the angular distribution of oriented bicelles is much narrower for the bicelles with smectic orientation, and, consequently, they keep aligned for much longer time (days) than those with nematic ordering (hours) outside the magnetic field. The understanding of the orientation/relaxation kinetics, as well as the morphologies of these "molecular goniometers" at molecular and supramolecular levels, allows controlling such an unprecedented long-range and long-lived smectic ordering of nanodisks and opens a wide field of applications for structural biology or material sciences.     

脑啡肽的固相合成与LC-MS/MS分离鉴定

多肽在生命过程中扮演着重要的角色,对其生理生化功能的研究与应用,离不开对单一多肽物质的需求,而化学合成法是获取目标多肽的最有效方法之一。对合成产物的分离与鉴定,是优化合成条件,以得到高产率的重要保证。以两种内源性神经肽亮氨酸脑啡肽和甲硫氨酸脑啡肽为模型,利用Fmoc固相多肽合成策略对其进行合成,并建立了HPLC-ESI-MS/MS新方法用于所制备的亮氨酸脑啡肽和甲硫氨酸脑啡肽的分离与结构鉴定。研究结果显示,主要合成产物均为目标多肽,副产物主要包括C端丢失1个氨基酸所形成的四肽,以及由于甲硫氨酸残基氧化而形成的含甲硫氨酸亚砜的多肽。该研究为高效合成含敏感氨基酸的生理活性多肽提供了新信息。     

Bilayer in small bicelles revealed by lipid-protein interactions using NMR spectroscopy

Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and small bicelles of dihexanoyl phosphatidylcholine (DHPC) and dimyristoyl phosphatidylcholine (DMPC) give insights into protein-lipid interactions. Intermolecular NOEs between hydrophobic tails of lipid and protein in the bicelles cover the surface area of OmpX forming a continuous cylindric jacket of approximately 2.7 nm in height. These NOEs originate only from DMPC molecules, and no NOEs from DHPC are observed. Further, these NOEs are mainly from methylene groups of the hydrophobic tails of DMPC, and only a handful of NOEs arise from methyl groups of the hydrophobic tails. The observed contacts indicate that the hydrophobic tails of DMPC are oriented parallel to the surface of OmpX and thus DMPC molecules form a bilayer in the vicinity of the protein. Thus, a bilayer exists in the small bicelles not only in the absence of but also in the presence of a membrane protein. In addition, the number of NOEs between the polar head groups of lipid molecules and protein is increased in the bicelles compared with those in micelles. This observation may be due to the closely packed head groups of the bilayer. Moreover, irregularity of hydrophobic interactions in the middle of the bilayer environment was observed. This observation together with the interactions between polar head groups and proteins gives a possible rationale for structural and functional differences of membrane proteins solubilized in micelles and in bilayer systems and hints at structural differences between protein-free and protein-loaded bilayers.     

Phospholipids Bilayers as Molecular Models for Drug- Membrane Interactions. The Case of the Antiepileptics Phenytoin and Carbamazepine

With the aim to better understand the molecular mechanisms of the interaction of phenytoin and carbamazepine with cell membranes we utilized a well-established model consisting in intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of its membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidyl-ethanolamine (DMPE), representative of phospholipid classes respectively located in the outer and inner monolayers of erythrocytes and other cell membranes. This report presents the following evidence that phenytoin and carbamazepine interact with membrane phospholipids: a) X-ray diffraction and fluorescence spectroscopy showed that both drugs preferentially interacted with DMPC; b) in IUM, the drugs induced a disordering effect on the polar head groups and acyl chains of the eryhrocyte membrane lipid bilayers; c) electron microscopy observations of human erythrocytes showed the echinocyte formation, an effect due to phenytoin and carbamazepine insertion in the outer monolayer of the red cell membrane.     

Micelle-induced curvature in a water-insoluble HIV-1 Env peptide revealed by NMR dipolar coupling measurement in stretched polyacrylamide gel

The structure of a water-insoluble fragment encompassing residues 282-304 of the HIV envelope protein gp41 is studied when solubilized by dihexanoyl phosphatidylcholine (DHPC) and by small bicelles, consisting of a 4:1 molar ratio of DHPC and dimyristoyl phosphatidylcholine (DMPC). Weak alignment with the magnetic field was accomplished in a stretched polyacrylamide gel, permitting measurement of one-bond (1)H-(15)N, (13)Ca-(13)C', and (13)C'-(15)N dipolar couplings, which formed the basis for determining the peptide structure. In both detergent systems, the peptide adopts an alpha-helical conformation from residue 4 through 18. In the presence of the DHPC micelles the helix is strongly curved towards the hydrophobic surface, whereas in the presence of bicelles a much weaker curvature in the opposite direction is observed.     

High-resolution 2D NMR spectroscopy of bicelles to measure the membrane interaction of ligands

Magnetically aligned bicelles are increasingly being used as model membranes in solution- and solid-state NMR studies of the structure, dynamics, topology, and interaction of membrane-associated peptides and proteins. These studies commonly utilize the PISEMA pulse sequence to measure dipolar coupling and chemical shift, the two key parameters used in subsequent structural analysis. In the present study, we demonstrate that the PISEMA and other rotating-frame pulse sequences are not suitable for the measurement of long-range heteronuclear dipolar couplings, and that they provide inaccurate values when multiple protons are coupled to a 13C nucleus. Furthermore, we demonstrate that a laboratory-frame separated-local-field experiment is capable of overcoming these difficulties in magnetically aligned bicelles. An extension of this approach to accurately measure 13C-31P and 1H-31P couplings from phospholipids, which are useful to understand the interaction of molecules with the membrane, is also described. In these 2D experiments, natural abundance 13C was observed from bicelles containing DMPC and DHPC lipid molecules. As a first application, these solid-state NMR approaches were utilized to probe the membrane interaction of an antidepressant molecule, desipramine, and its location in the membrane.     

Diffusion of PEG confined between lamellae of negatively magnetically aligned bicelles: pulsed field gradient 1H NMR measurements

The diffusion of various molecular weight poly(ethyleneglycol)s (PEG) confined between the lamellae of magnetically aligned bicelles has been measured using stimulated echo (STE) pulsed field gradient (PFG) 1H nuclear magnetic resonance (NMR) spectroscopy. Bicelles were formulated to contain dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), and dihexanoylphosphatidylcholine (DHPC) in the proportion DMPG/DMPC = 0.05 and q = (DMPC + DMPG)/DHPC = 4.5. PEG diffusion within the interlamellar spaces between such bicelles was found to be unrestricted over diffusion distances of tens of microns. Two confinement regimes could be differentiated according to the dependence of the reduced PEG diffusivity D/D0, where D0 is the unconfined PEG diffusion coefficient, on the relative confinement Rh/H, where Rh is the unperturbed hydration radius of the particular PEG and H approximately 60 A is the separation between apposing lamellae of the magnetically aligned bicelles. In the regime Rh/H < 0.4, the reduced PEG diffusivity was altered only in proportion to the viscosity increase associated with the bicelle dispersion relative to bulk solution. In the regime Rh/H > 0.4, the reduced PEG diffusivity scaled as (Rh/H)-2/3, in agreement with scaling theories for confined polymers.     

An alternative method for pucker determination in carbohydrates from residual dipolar couplings: a solution NMR study of the fructofuranosyl ring of sucrose

A simple solution NMR method is presented for pucker determination of five-membered rings using only residual dipolar couplings obtained in a single liquid crystalline medium, DMPC/DHPC bicelles (DMPC = dimyristoylphosphatidylcholine; DHPC = dihexanoylphosphatidylcholine). The method was applied to determine the pucker of the fructofuranosyl ring of sucrose. The results indicate a fructofuranosyl pucker phase in the 20 degrees - 70 degrees range. The pucker phases are in agreement with those from previous NMR and optical spectroscopic studies and, importantly, do not rely on empirically parametrized Karplus curves. Furthermore, the analysis implies more than one stable pucker phase and rapid ring interconversion in this range. The present results suggest that using residual dipolar couplings alone can reveal multiple conformations present in solution. Hence, when a sufficient number of residual dipolar coupling constants is measured, the outcome is a robust, reliable, and independent route for determining carbohydrate and nucleic acid structure by NMR spectroscopy.     

Lipid modifications of a Ras peptide exhibit altered packing and mobility versus host membrane as detected by 2H solid-state NMR

The human N-ras protein binds to cellular membranes by insertion of two covalently bound posttranslational lipid modifications, which is crucial for its function in signal transduction and cell proliferation. Mutations in ras may lead to unregulated cell growth and eventually cancer, making it an important therapeutic target. Here we have investigated the molecular details of the membrane binding mechanism. A heptapeptide derived from the C-terminus of the human N-ras protein was synthesized including two hexadecyl modifications. Solid-state 2H NMR was used to determine the packing and molecular dynamics of the ras lipid chains as well as the phospholipid matrix. Separately labeling the chains of the peptide and the phospholipids with 2H enabled us to obtain atomically resolved parameters relevant to their structural dynamics. While the presence of ras only marginally affected the packing of DMPC membranes, dramatically lower order parameters (S(CD)) were observed for the ras acyl chains indicating modified packing properties. Essentially identical projected lengths of the 16:0 ras chains and the 14:0 DMPC chains were found, implying that the polypeptide backbone is located at the lipid-water interface. Dynamical properties of both the ras and phospholipid chains were determined from spin-lattice 2H relaxation (R1Z) measurements. Plots of R1Z rates versus the corresponding squared segmental order parameters revealed striking differences. We propose the ras peptide is confined to microdomains containing DMPC chains which are in exchange with the bulk bilayer on the 2H NMR time scale (approximately 10(-5) s). Compared to the host DMPC matrix, the ras lipid modifications are extremely flexible and undergo relatively large amplitude motions. It is hypothesized that this flexibility is a requirement for the optimal anchoring of lipid-modified proteins to cellular membranes.     

Cholesterol increases the magnetic aligning of bicellar disks from an aqueous mixture of DMPC and DMPE-DTPA with complexed thulium ions

Aqueous mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-diethylenetriamine pentaacetate (DMPE-DTPA) with complexed thulium ions (Tm(3+)), and cholesterol with varying molar ratio were studied at different temperatures in the presence and absence of a magnetic field. For mixtures without cholesterol weakly magnetically alignable small disks, so-called bicelles, are formed at temperatures below the phase transition temperature (5-22 °C), as shown by cryo-transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS). In presence of 16 mol % cholesterol the disk size and the magnetic alignability were larger within the entire temperature range studied (5-40 °C). Cholesterol acts as a spacer between DMPE-DTPA with complexed Tm(3+), allowing these molecules to integrate more frequently into the planar part of the bicelles. Replacing DMPC partially by cholesterol thus lead to an increase in magnetic aligning by a higher amount of the magnetic handles (Tm(3+) complexed to DMPE-DTPA) in the plane and by an increased number of phospholipids in the enlarged bicelles. The magnetic aligning was most pronounced at 5 °C. The temperature-dependent structural changes of the DMPC/cholesterol/DMPE-DTPA/Tm(3+) aqueous mixtures are complex, including the transient appearance of holes in the disks at intermediate temperatures.     

The cell-penetrating peptide TAT(48-60) induces a non-lamellar phase in DMPC membranes.

Cell-penetrating peptides (CPPs) are short polycationic sequences that can translocate into cells without disintegrating the plasma membrane. CPPs are useful tools for delivering cargo, but their molecular mechanism of crossing the lipid bilayer remains unclear. Here we study the interaction of the HIV-derived CPP TAT (48-60) with model membranes by solid-state NMR spectroscopy and electron microscopy. The peptide induces a pronounced isotropic (31)P NMR signal in zwitterionic DMPC, but not in anionic DMPG bilayers. Octaarginine and to a lesser extent octalysine have the same effect, in contrast to other cationic amphiphilic membrane-active peptides. The observed non-lamellar lipid morphology is attributed to specific interactions of polycationic peptides with phosphocholine head groups, rather than to electrostatic interactions. Freeze-fracture electron microscopy indicates that TAT(48-60) induces the formation of rodlike, presumably inverted micelles in DMPC, which may represent intermediates during the translocation across eukaryotic membranes.     

Residual dipolar coupling measurements of transmembrane proteins using aligned low-q bicelles and high-resolution magic angle spinning NMR spectroscopy

Bicelles are a major medium form to produce weak alignment of soluble proteins for residual dipolar coupling (RDC) measurements. The obstacle to using the same type of bicelles for transmembrane proteins with solution-state NMR spectroscopy is the loss of signals due to the adhesion or penetration of the proteins into large bicelles, resulting in slow protein tumbling. In this study, weak alignment of the second and third transmembrane domains (TM23) of the human glycine receptor (GlyR) was achieved in low-q bicelles (q = DMPC/DHPC). Although protein-free bicelles with such low q would likely show isotropic properties, the insertion of TM23 induced weakly preferred orientations so that the RDC of the embedded protein can be measured. The extent of the alignment increased but the TM23 signal intensity decreased when q was varied from 0.19 to 0.60. A q of 0.50 was found to be an optimal compromise between alignment and the signal-to-noise ratio. In each pair of NMR experiments for RDC measurements, the same sample and pulse sequence were used, with one being performed at high-resolution magic-angle spinning to obtain pure J-couplings without RDC. A meaningful structure refinement in bicelles was possible by iteratively fitting the experimental RDCs to the back-calculated RDCs using the high-resolution NMR structure of GlyR TM23 in trifluoroethanol as the starting template. Combination of this method with the conventional high-resolution NMR in membrane mimicking mixtures of water and organic solvents offers an attractive way to derive structural information for membrane proteins in their native environment.     

A high-resolution solid-state NMR approach for the structural studies of bicelles

Bicelles are increasingly being used as membrane mimicking systems in NMR experiments to investigate the structure of membrane proteins. In this study, we demonstrate the effectiveness of a 2D solid-state NMR approach that can be used to measure the structural constraints, such as heteronuclear dipolar couplings between 1H, 13C, and 31P nuclei, in bicelles without the need for isotopic enrichment. This method does not require a high radio frequency power unlike the presently used rotating-frame separated-local-field (SLF) techniques, such as PISEMA. In addition, multiple dipolar couplings can be measured accurately, and the presence of a strong dipolar coupling does not suppress the weak couplings. High-resolution spectra obtained from magnetically aligned DMPC:DHPC bicelles even in the presence of peptides suggest that this approach will be useful in understanding lipid-protein interactions that play a vital role in shaping up the function of membrane proteins.     

Remarkably enhanced inhibitory effects of three-component hybrid liposomes including sugar surfactants on the growth of lung carcinoma cells

Sugar parts play important roles in recognizing molecules on the cell membranes. We successfully produced sugar-type micellar surfactants, lactonoalkylamide (LacCn), for the first time. Spherical vesicles, three-component hybrid liposomes, were obtained after the sonication of the mixture of L-alpha-dimyristoyl-phosphatidylcholine (DMPC), Tween 20 and LacCn (DMPC:Tween 20:LacCn=65:7:28). It is noteworthy that high inhibitory effects of the three-component hybrid liposomes composed of DMPC, Tween 20, and LacCn (DMPC:Tween 20:LacCn=65:7:28) on the growth of glioma (U251) and lung adenocarcinoma (RERF-LC-OK) cells were attained in vitro without any drug, although no significant inhibitory effects of any individual component (DMPC, Tween 20, LacCn) or the two-component hybrid liposomes of DMPC and Tween 20 on the growth of tumor cells examined were obtained.     

Bicellar systems as modifiers of skin lipid structure

The characterization of different bicellar aggregates and the effects of these systems on the stratum corneum (SC) microstructure have been studied. Dynamic light scattering (DLS) and freeze fracture electron microscopy (FFEM) techniques showed that both of the systems studied, dimyristoyl-phosphatidylcholine/dihexanoyl-phosphocholine (DMPC/DHPC) and dipalmitoyl-phosphocholine (DPPC)/DHPC, were formed by small discoidal aggregates at room temperature (20°C). Treating skin with DMPC/DHPC bicelles does not affect the SC lipid microstructure, whereas bicellar systems formed by DPPC and DHPC can promote the formation of new structures in the SC lipid domains. This indicates the passage of lipids from bicelles through the SC layers and also a possible interaction of these lipids with the SC lipids. Given the absence of surfactant in the bicellar composition and the small size of these structures, the use of these smart nano-systems offers great advantages over other lipid systems for dermatological purposes. Bicelles could be promising applications as drug carriers through the skin. This contribution, based on the new biological use of bicelles, may be useful to scientists engaged in colloid science and offers a new tool for different applications in skin and cosmetic research.     

Glycopeptides related to beta-endorphin adopt helical amphipathic conformations in the presence of lipid bilayers

A series of glycosylated endorphin analogues designed to penetrate the blood-brain barrier (BBB) have been studied by circular dichroism and by 2D-NMR in the presence of water; TFE/water; SDS micelles; and in the presence of both neutral and anionic bicelles. In water, the glycopeptides showed only nascent helix behavior and random coil conformations. Chemical shift indices and nuclear Overhauser effects (NOE) confirmed helices in the presence of membrane mimics. NOE volumes provided distance constraints for molecular dynamics calculations used to provide detailed backbone conformations. In all cases, the glycopeptides were largely helical in the presence of membrane bilayer models (micelles or bicelles). Plasmon waveguide resonance (PWR) studies showed hen egg phosphatidyl choline (PC) bilayers produce amphipathic helices laying parallel to the membrane surface, with dissociation constants (K(D)) in the low nanomolar to micromolar concentration range. Two low-energy states are suggested for the glycosylated endorphin analogues, a flexible aqueous state and a restricted membrane bound state. Strong interactions between the glycopeptide amphipaths and membranes are crucial for penetration of the BBB via an endocytotic mechanism (transcytosis).     

NMR methods for studying the structure and dynamics of oncogenic and antihistaminic peptides in biomembranes

We present several applications of both wide-line and magic angle spinning (MAS) solid-state NMR of bicelles in which are embedded fragments of a tyrosine kinase receptor or enkephalins. The magnetically orientable bicelle membranes are shown to be of particular interest for studying the functional properties of lipids and proteins in a state that is very close to their natural environment. Quadrupolar, dipolar and chemical shielding interactions can be used to determine minute alterations of internal membrane dynamics and the orientation of peptides with respect to the membrane plane. MAS of bicelles can in turn lead to high-resolution proton spectra of hydrated membranes. Using deuterium-proton contrast methods one can then obtain pseudo-high-resolution proton spectra of peptides or proteins embedded in deuterated membranes and determine their atomic 3D structure using quasi-conventional liquid-state NMR methods.     

Specific binding structures of dendrimers on lipid bilayer membranes

Dissipative particle dynamics simulations are used to study the specific binding structures of polyamidoamine (PAMAM) dendrimers on amphiphilic membranes and the permeation mechanisms. Mutually consistent coarse-grained (CG) models both for PAMAM dendrimers and for dimyristoylphosphatidylcholine (DMPC) lipid molecules are constructed. The PAMAM CG model describes correctly the conformational behavior of the dendrimers, and the DMPC CG model can properly give the surface tension of the amphiphilic membrane. A series of systematic simulations is performed to investigate the binding structures of the dendrimers on membranes with varied length of the hydrophobic tails of amphiphiles. The permeability of dendrimers across membranes is enhanced upon increasing the dendrimer size (generation). The length of the hydrophobic tails of amphiphiles in turn affects the dendrimer conformation, as well as the binding structure of the dendrimer-membrane complexes. The negative curvature of the membrane formed in the dendrimer-membrane complexes is related to dendrimer concentration. Higher dendrimer concentration together with increased dendrimer generation is observed to enhance the permeability of dendrimers across the amphiphilic membranes.