Preparation Of Lipid Bilayers Research Articles

Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples

Solid-state NMR spectroscopy has been used successfully for characterizing the structure and dynamics of membrane proteins as well as their interactions with other proteins in lipid bilayers. Such an environment is often necessary for achieving native-like structures. Sample preparation is the key to this success. Here we present a detailed description of a robust protocol that results in high-quality membrane protein samples for both magic-angle spinning and oriented-sample solid-state NMR. The procedure is demonstrated using two proteins: CrgA (two transmembrane helices) and Rv1861 (three transmembrane helices), both from Mycobacterium tuberculosis. The success of this procedure relies on two points. First, for samples for both types of NMR experiment, the reconstitution of the protein from a detergent environment to an environment in which it is incorporated into liposomes results in 'complete' removal of detergent. Second, for the oriented samples, proper dehydration followed by rehydration of the proteoliposomes is essential. By using this protocol, proteoliposome samples for magic-angle spinning NMR and uniformly aligned samples (orientational mosaicity of <1°) for oriented-sample NMR can be obtained within 10 d.

Binding of MgtR, a Salmonella Transmembrane Regulatory Peptide, to MgtC, a Mycobacterium tuberculosis Virulence Factor: A Structural Study

MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium, inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologous to its S. Typhi analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhi MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.

Effect of Lipid Bilayer Composition on the Membrane-Binding Ability and Depth of Insertion of Antimicrobial Piscidin 1: A Solid-State Deuterium NMR Study

Piscidins constitute a family of amphipathic, cationic, antimicrobial peptides (AMPs) found in the skin, gills and gastrointestinal tracts of hybrid striped bass. Unstructured in water, they become alpha-helical in the presence of phospholipids. It is believed that their ability to disrupt phospholipid bilayers is related to their antimicrobial effects on yeasts and bacteria. Interestingly, the minimum inhibitory concentrations of piscidin tend to be higher for yeasts than most bacteria. Here, we used Circular Dichroism (CD) and deuterium solid-state Nuclear Magnetic Resonance (ssNMR) to investigate the membrane-binding ability and depth of insertion of piscidin 1 in different membrane mimetic systems. Aligned lipid bilayer preparations were made using 3:1 POPC/POPG-d31 (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol-d31) and 3:1 POPC/POPS-d31 (1-palmitoyl 2-oleoyl phosphatidylserine-d31) to mimic bacterial and yeast cell membranes, respectively. To test the effects of ergosterol, a sterol found in yeasts, we added 20% ergosterol (molar ratio) to the lipid mixtures. We hypothesized that both PS and ergosterol would reduce the ability of piscidin to bind and disrupt bilayers.Deuterium quadrupolar splittings, which were obtained for deuterons in the methylene and terminal methyl groups of the phospholipid acyl chains, were used to assess the disordering effect of piscidin on the acyl chains. Our results suggest that the type of anionic lipid component (e.g., PG versus PS) does not affect acyl chain disordering by piscidin while ergosterol appears to decrease its disordering effect. The data also give evidence that membrane-bound piscidin is located close to the phospholipid headgroups. Overall, these studies help us gain some insight into specific physicochemical features of lipid membranes that affect piscidin-lipid interactions. The implications in terms of piscidin's mode of action will be discussed.

Ryanodine Receptor Phosphorylation by Calcium/Calmodulin-Dependent Protein Kinase II Promotes Life-Threatening Ventricular Arrhythmias in Mice With Heart Failure

approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca(2+) remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. mice in which the S2814 Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca(2+) release events, resulting in reduced sarcoplasmic reticulum Ca(2+) load on confocal microscopy. These Ca(2+) release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. our results suggest that Ca(2+)/calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca(2+) release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.

Influence of Mono- And Divalent Ions on the Formation of Supported Phospholipid Bilayers via Vesicle Adsorption

We have used the quartz crystal microbalance with dissipation monitoring (QCM-D) technique to investigate how mono- and divalent cations influence the formation of supported (phospho)lipid bilayers (SPB, SLB), occurring via deposition of nanosized palmitoyloleoyl phosphatidylcholine (POPC) vesicles on a SiO2 support. This process is known to proceed via initial adsorption of intact vesicles until a critical surface coverage is reached, where the combination of vesicle-surface and vesicle-vesicle interaction causes the vesicles to rupture. New vesicles then rupture and the lipid fragments fuse until a final continuous bilayer is formed. We have explored how this process and the critical coverage are influenced by different mono- and divalent ions and ion concentrations, keeping the anions the same throughout the experiments. The same qualitative kinetics is observed for all cations. However, different ions cause quite different quantitative kinetics. When compared with monovalent ions, even very small added concentrations of divalent cations cause a strong reduction of the critical coverage, where conversion of intact, adsorbed vesicles to bilayer occurs. This bilayer promoting effect increases in the order Sr2+<Ca2+<Mg2+. Monovalent cations exhibit a much weaker but similar effect in the order Li+>Na+>K+. The results are of practical value for preparation of lipid bilayers and help shed light on the role of ions and on electrostatic effects at membrane surfaces/interfaces.

The design of molecular sensing interfaces with lipid-bilayer assemblies

We describe the current status of techniques for preparing planar lipid bilayers, the fusion of sensory elements and the design of bilayer interfaces for analytical applications. Advances in bilayer fabrication have allowed preparation of lipid bilayers with membrane resistance of >1 GΩ (“gigaseal”) in flow, microfluidic and array formats, which have enabled single-channel and multi-channel recording. Not only biological but also engineered channels have been adopted as sensory elements for lipid-bilayer-based biosensors. Synthetic and inorganic channels are also emerging for designing membranes for lipid-bilayer sensors. We discuss the potential of lipid bilayers containing biological, engineered or synthetic channels for the design of biosensors, including drug-screening biosensors.

Structural biology of transmembrane domains: Efficient production and characterization of transmembrane peptides by NMR

Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins.

Structure and distribution of the Bacillus thuringiensis Cry4Ba toxin in lipid membranes

Bacillus thuringiensis Cry δ-endotoxins cause death of susceptible insect larvae by forming lytic pores in the midgut epithelial cell membranes. The 65 kDa trypsin activated Cry4Ba toxin was previously shown to be capable of permeabilizing liposomes and forming ionic channels in receptor-free planar lipid bilayers. Here, magnetic ACmode (MACmode) atomic force microscopy (AFM) was used to characterize the lateral distribution and the native molecular structure of the Cry4Ba toxin in the membrane. Liposome fusion and the Langmuir–Blodgett technique were employed for supported lipid bilayer preparations. The toxin preferentially inserted in a self-assembled structure, rather than as a single monomeric molecule. In addition, the spontaneous insertion into receptor-free lipid bilayers lead to formation of characteristic pore-like structures with four-fold symmetry, suggesting that tetramers are the preferred oligomerization state of this toxin.

Modulating dipoles for structure-function correlations in the gramicidin A channel.

Dipoles of the tryptophan indole side chains have a direct impact on ion conductance in the gramicidin channel. Here, fluorination of the indoles (both 5- and 6-fluoro) is used to manipulate both the orientations and the magnitudes of the dipoles. The orientations and positions with respect to the channel axis were determined using (2)H solid state NMR of uniformly aligned lipid bilayer preparations. By exchange of the remaining four protons in the indole ring for deuterium, comparison could be made to d(5)-indole spectra that have previously been recorded for each of the four indoles of gramicidin A. After making the assignments which were aided by the observation of (19)F-(2)H dipolar interactions, we found that fluorination caused only minor changes in side chain conformation. With the high-resolution structural characterization of the fluorinated indoles in position 11, 13, and 15, the electrostatic interactions with a cation at the channel and bilayer center can be predicted and the influence of the modified dipoles on ion conductance estimated. The importance of the long-range electrostatic interaction was recently documented with the observation of alpha-helical dipoles oriented toward the bilayer center on the ion conductance pathway for the Streptomyces K(+) channel. We present direct measurements of the orientation of gramicidin channel F-Trp positions for use in analysis of dipole effects on channel permeation.

Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis

Small angle X-ray diffraction was used to examine arterial smooth muscle cell (SMC) plasma membranes isolated from control and cholesterol-fed (2%) atherosclerotic rabbits. A microsomal membrane enriched with plasma membrane obtained from animals fed cholesterol for up to 13 weeks showed a progressive elevation in the membrane unesterified (free) cholesterol:phospholipid (C/PL) mole ratio. Beyond 9 weeks of cholesterol feeding, X-ray diffraction patterns demonstrated a lateral immiscible cholesterol domain at 37 degrees C with a unit cell periodicity of 34 A coexisting within the liquid crystalline lipid bilayer. On warming, the immiscible cholesterol domain disappeared, and on cooling it reappeared, indicating that the immiscible cholesterol domain was fully reversible. These effects were reproduced in a model C/PL binary lipid system. In rabbits fed cholesterol for less than 9 weeks, lesser increases in membrane C/PL mole ratio were observed. X-ray diffraction analysis demonstrated an increase in membrane bilayer width that correlated with the C/PL mole ratio. This effect was also reproduced in a C/PL binary lipid system. Taken together, these findings demonstrate that in vivo, feeding of cholesterol causes cholesterol-phospholipid interactions in the membrane bilayer that alter bilayer structure and organization. This interaction results in an increase in bilayer width peaking at a saturating membrane cholesterol concentration, beyond which lateral phase separation occurs resulting in the formation of separate cholesterol bilayer domains. These alterations in structure and organization in SMC plasma membranes may have significance in phenotypic modulation or aortic SMC during early atherogenesis.

Monovalent cation transport: lack of structural deformation upon cation binding.

Cations often deform the structure of regulatory proteins to affect a functional response, but for other protein functions a more passive effect is desired. For instance, it is shown here that in the conductance of Na+ by the gramicidin channel there appears to be no significant structural deformation of either the side chains or backbone upon Na+ binding in the channel. This is based on 15N and 13C chemical shifts, 2H quadrupolar interactions, and 15N-2H dipolar interactions obtained by solid-state NMR spectroscopy of uniformly aligned lipid bilayer preparations of the gramicidin channel in the presence and absence of Na+. This conclusion is despite some significant changes in the 15N alpha and 13C1 chemical shift values which are argued here to be the result of indirect polarization effects upon cation binding rather than reflections of structural and dynamic changes. The lack of structural deformation implies that Na+ moves to the carbonyl oxygens lining the pore of this channel for solvation rather than the carbonyl groups moving in toward the channel axis. This forces the cations onto a helical path following the positions of the carbonyl oxygens around the channel pore. Furthermore, an ideal binding site geometry for Na+ in the channel is avoided. Instead, adequate binding energy is provided by the channel to compensate for the loss of hydration energy when the cations enter the channel. The avoidance of strong binding ensures that efficient transport of the cations through the channel can be realized.

Tryptophan dynamics and structural refinement in a lipid bilayer environment: solid state NMR of the gramicidin channel.

Tryptophans in the gramicidin channel are important for defining the conformation and the orientation with respect to the bilayer normal and for facilitating cation conductance. Here, high-resolution structure and dynamics of these rings are characterized by solid state NMR. Both oriented and unoriented lipid bilayer preparations are used. Fast frozen lipid bilayer preparations of unoriented samples have been used to obtain static characterizations of nuclear spin interaction tensors. The temperature dependence of these unoriented samples and the spectral features of fast frozen oriented samples were used to experimentally define the local motions of the individual indole rings. Local motions were shown to have amplitudes as high as +/- 29 degrees, and the motions were dominated by libration about the chi 2 axis. The high-resolution structure has been achieved by interpreting seven precise (+/- 0.3 degree) orientational constraints from 2H and 15N NMR for each indole ring in light of the motionally averaged interaction tensors. Each of the four indoles is restricted to a unique orientation of the ring with respect to the bilayer normal and one of two possible rotameric states. The side chain torsion angles for each residue are very similar, generating similar electric dipole moment orientations with respect to the channel.