Proteins In Lipid Bilayers Research Articles

Curvature-dependent protein-lipid bilayer interaction and cell mechanosensitivity.

Cells respond to applied external forces through different mechanosensitive processes, with many of them based on the interaction between membrane-embedded proteins and their lipid environments. This interaction can depend on membrane curvature at the location of such proteins. Here we elucidate the general characteristics of a macroscopically defined protein-lipid bilayer interaction based on a mismatch between the shape of the protein surface and the surrounding membrane curvature. It is shown how the parameters of this interaction are related to the experimentally determined distribution of membrane-embedded proteins between highly curved tubular and flat membrane regions of a giant phospholipid vesicle. The results obtained for such distribution of potassium channel KvAP are presented as an example. Possible participation of the curvature-dependent protein-lipid bilayer interaction in mechanosensitive processes is indicated.

Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces.

We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.

Molecular dynamics simulations of the bacterial UraA H+-uracil symporter in lipid bilayers reveal a closed state and a selective interaction with cardiolipin.

The Escherichia coli UraA H+-uracil symporter is a member of the nucleobase/ascorbate transporter (NAT) family of proteins, and is responsible for the proton-driven uptake of uracil. Multiscale molecular dynamics simulations of the UraA symporter in phospholipid bilayers consisting of: 1) 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC); 2) 1-palmitoyl 2-oleoyl-phosphatidylethanolamine (POPE); and 3) a mixture of 75% POPE, 20% 1-palmitoyl 2-oleoyl-phosphatidylglycerol (POPG); and 5% 1-palmitoyl 2-oleoyl-diphosphatidylglycerol/cardiolipin (CL) to mimic the lipid composition of the bacterial inner membrane, were performed using the MARTINI coarse-grained force field to self-assemble lipids around the crystal structure of this membrane transport protein, followed by atomistic simulations. The overall fold of the protein in lipid bilayers remained similar to the crystal structure in detergent on the timescale of our simulations. Simulations were performed in the absence of uracil, and resulted in a closed state of the transporter, due to relative movement of the gate and core domains. Anionic lipids, including POPG and especially CL, were found to associate with UraA, involving interactions between specific basic residues in loop regions and phosphate oxygens of the CL head group. In particular, three CL binding sites were identified on UraA: two in the inner leaflet and a single site in the outer leaflet. Mutation of basic residues in the binding sites resulted in the loss of CL binding in the simulations. CL may play a role as a “proton trap” that channels protons to and from this transporter within CL-enriched areas of the inner bacterial membrane.

Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5-0.3 ppm for (13)C line widths and <0.5 ppm (15)N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via (1)H detected MAS NMR.

PH-Dependent Conformational Changes in the Influenza a M2 Full-Length Protein in Lipid Bilayers

Influenza A M2 protein is a homo-tetrameric, membrane protein that conducts protons as one of its multiple functions. Monomeric M2 has an N-terminus that appears to have some b-strand and cysteine disulfide linkages, single transmembrane helix followed by an amphipathic helix and C-terminus whose structure is largely unknown. Together, the TM and amphipathic helices are called ‘the conductance domain”. Even though the conductance domain structure is solved in lipid bilayers, the structure of the full-length protein is not yet fully characterized although considerable spectroscopy of the protein has been published showing that the transmembrane helix within the full length protein is similar to that in the conductance domain.Although, the effect of pH on different truncated versions of the M2 protein has been observed, its effect on the full-length M2 in lipid bilayers is not yet reported. Here, we present how pH affects the opening of the pore in the full-length protein in lipid bilayers and on amantadine block of the wild type M2 channel at pH values that correspond to open and closed states of the channel. These changes in the backbone conformation were determined by unambiguous, inter-monomer distance measurements using different amino acid specific labeled monomers and solid state NMR spectroscopy. In addition, data obtained from the drug resistant mutant (S31N full length) is presented.Distances between the monomers to date have been sparse - these unambiguous restraints not only enhance the structure of the protein surrounding the aqueous pore through the membrane, but in particular there have been no high resolution distance restraints associated with the amphipathic helix until now. Here, we validate the orientation of the amphipathic helix using unambiguous, inter-monomer distance measurements. Effects of environment/conditions (cholesterol/pH) on these measurements will also presented.

Glass: A Multi-Platform Specimen Supporting Substrate for Precision Single Molecule Studies of Membrane Proteins

High resolution (≈1 nm lateral resolution) biological AFM imaging has been carried out almost exclusively using freshly cleaved mica as a specimen supporting surface, but mica suffers from a fundamental limitation that has hindered AFM's broader integration with many modern optical methods. Mica exhibits biaxial birefringence; indeed, this naturally occurring material is used commercially for constructing optical wave plates. In general, propagation through birefringent material alters the polarization state and bifurcates the propagation direction of light in a manner which varies with thickness. This makes it challenging to incorporate freshly cleaved mica substrates with modern optical methods, many of which employ highly focused and polarized laser beams passing through then specimen plane. Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on common microscope cover glass following a straight-forward cleaning procedure. Direct comparison between data obtained on glass and on mica show no significant differences in AFM image fidelity. This work opens the door for combining high resolution biological AFM with powerful optical methods that require optically isotropic substrates such as ultra-stable1 and direct 3D AFM2. In turn, this capability should enable long timescale conformational dynamics measurements of membrane proteins in near-native conditions. 1.King, G.M., Carter, A.R., Churnside, A.B., Eberle, L.S. & Perkins, T.T. Ultrastable atomic force microscopy: atomic-scale stability and registration in ambient conditions. Nano Letters 9, 1451 (2009). 2.Sigdel, K.P., Grayer, J.S. & King, G.M. Three-dimensional atomic force microscopy: interaction force vector by direct observation of tip trajectory. Nano Lett 13, 5106-5111 (2013).

Development of the field of structural physiology.

Electron crystallography is especially useful for studying the structure and function of membrane proteins — key molecules with important functions in neural and other cells. Electron crystallography is now an established technique for analyzing the structures of membrane proteins in lipid bilayers that closely simulate their natural biological environment. Utilizing cryo-electron microscopes with helium-cooled specimen stages that were developed through a personal motivation to understand the functions of neural systems from a structural point of view, the structures of membrane proteins can be analyzed at a higher than 3 Å resolution. This review covers four objectives. First, I introduce the new research field of structural physiology. Second, I recount some of the struggles involved in developing cryo-electron microscopes. Third, I review the structural and functional analyses of membrane proteins mainly by electron crystallography using cryo-electron microscopes. Finally, I discuss multifunctional channels named “adhennels” based on structures analyzed using electron and X-ray crystallography.

Sorting of integral membrane proteins mediated by curvature-dependent protein-lipid bilayer interaction.

Cell membrane proteins, both bound and integral, are known to preferentially accumulate at membrane locations with curvatures favorable to their shape. This is mainly due to the curvature dependent interaction between membrane proteins and their lipid environment. Here, we analyze the effects of the protein-lipid bilayer interaction energy due to mismatch between the protein shape and the principal curvatures of the surrounding bilayer. The role of different macroscopic parameters that define the interaction energy term is elucidated in relation to recent experiment in which the lateral distribution of a membrane embedded protein potassium channel KvAP is measured on a giant unilamellar lipid vesicle (reservoir) and a narrow tubular extension - a tether - kept at constant length. The dependence of the sorting ratio, defined as the ratio between the areal density of the protein on the tether and on the vesicle, on the inverse tether radius is influenced by the strength of the interaction, the intrinsic shape of the membrane embedded protein, and its abundance in the reservoir. It is described how the values of these constants can be extracted from experiments. The intrinsic principal curvatures of a protein are related to the tether radius at which the sorting ratio attains its maximum value. The estimate of the principal intrinsic curvature of the protein KvAP, obtained by comparing the experimental and theoretical sorting behavior, is consistent with the available information on its structure.

Integration and Oligomerization of Bax Protein in Lipid Bilayers Characterized by Single Molecule Fluorescence Study

Bax is a pro-apoptotic Bcl-2 family protein. The activated Bax translocates to mitochondria, where it forms pore and permeabilizes the mitochondrial outer membrane. This process requires the BH3-only activator protein (i.e. tBid) and can be inhibited by anti-apoptotic Bcl-2 family proteins such as Bcl-xL. Here by using single molecule fluorescence techniques, we studied the integration and oligomerization of Bax in lipid bilayers. Our study revealed that Bax can bind to lipid membrane spontaneously in the absence of tBid. The Bax pore formation undergoes at least two steps: pre-pore formation and membrane insertion. The activated Bax triggered by tBid or BH3 domain peptide integrates on bilayers and tends to form tetramers, which are termed as pre-pore. Subsequent insertion of the pre-pore into membrane is highly dependent on the composition of cardiolipin in lipid bilayers. Bcl-xL can translocate Bax from membrane to solution and inhibit the pore formation. The study of Bax integration and oligomerization at the single molecule level provides new evidences that may help elucidate the pore formation of Bax and its regulatory mechanism in apoptosis.

Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration.

Recently, the recombinant 30Kc19 protein, originating from silkworm hemolymph of Bombyx mori has attracted attention due to its cell-penetrating property and potential application as a protein delivery system. However, this observation of penetration across cell membrane has raised questions concerning the interaction of the protein-lipid bilayer. Here, we report a dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moieties; sodium dodecyl sulfate (SDS) or phospholipid. Native PAGE showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. In the glutathione-S-transferase (GST) pull-down assay, supplementation of the 30Kc19 protein to mammalian cell culture medium showed dimerization and penetration; due to phospholipids at the cell membrane, the main components of the lipid bilayer. Mutagenesis was performed, and penetration was observed by 30Kc19 C76A and not 30Kc19 C57A, which meant that the presence of cysteine at position 57 (Cys-57) is involved in dimerization of the 30Kc19 at the cell membrane during penetration. We anticipate application of the native 30Kc19 protein with high cell-penetrating efficiency for delivery of cargos to various cell types. The intracellular cargo delivery using the 30Kc19 protein is a non-virus derived (e.g. TAT) delivery method, which can open up new approaches for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.

Solid-state NMR shows that dynamically different domains of membrane proteins have different hydration dependence.

Hydration has a profound influence on the structure, dynamics, and functions of membrane and membrane-embedded proteins. So far the hydration response of molecular dynamics of membrane proteins in lipid bilayers is poorly understood. Here, we reveal different hydration dependence of the dynamics in dynamically different domains of membrane proteins by multidimensional magic angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy using 121-residue integral diacylglycerol kinase (DAGK) in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) lipid bilayers as a model system. The highly mobile and immobile domains of DAGK and their water accessibilities are identified site-specifically by scalar- and dipolar-coupling based MAS ssNMR experiments, respectively. Our experiments reveal different hydration dependence of the dynamics in highly mobile and immobile domains of membrane proteins. We demonstrate that the fast, large-amplitude motions in highly mobile domains are not triggered until 20% hydration, enhanced at 20-50% hydration and unchanged at above 50% hydration. In contrast, motions on submicrosecond time scale of immobile residues are observed to be independent of the hydration levels in gel phase of lipids, and at the temperature near gel-liquid crystalline phase transition, amplitude of whole-molecule rotations around the bilayer normal is dominated by the fluidity of lipid bilayers, which is strongly hydration dependent. The hydration dependence of the dynamics of DAGK revealed by this study provides new insights into the correlations of hydration to dynamics and function of membrane proteins in lipid bilayers.

Frame‐Guided Assembly of Vesicles with Programmed Geometry and Dimensions

In molecular self-assembly molecules form organized structures or patterns. The control of the self-assembly process is an important and challenging topic. Inspired by the cytoskeletal-membrane protein lipid bilayer system that determines the shape of eukaryotic cells, we developed a frame-guided assembly process as a general strategy to prepare heterovesicles with programmed geometry and dimensions. This method offers greater control over self-assembly which may benefit the understanding of the formation mechanism as well as the functions of the cell membrane.

Frame‐Guided Assembly of Vesicles with Programmed Geometry and Dimensions

AbstractIn molecular self‐assembly molecules form organized structures or patterns. The control of the self‐assembly process is an important and challenging topic. Inspired by the cytoskeletal‐membrane protein lipid bilayer system that determines the shape of eukaryotic cells, we developed a frame‐guided assembly process as a general strategy to prepare heterovesicles with programmed geometry and dimensions. This method offers greater control over self‐assembly which may benefit the understanding of the formation mechanism as well as the functions of the cell membrane.

NMR-Restrained Protein Structure Calculations in Implicit Environment

The aqueous and lipid membrane environments that make up the intra- and extra-cellular compartments of biological organisms are critical for supporting the structural and functional integrity of both soluble proteins and membrane proteins. NMR spectroscopy is extremely versatile and adept at characterizing the structures and functions of both types of proteins in samples that closely resemble the native environment. However, structure calculations in explicit solvent or explicit lipids are computationally expensive. Therefore, even when NMR structural restraints are measured in a native-like protein environment, protein structures are typically calculated using simple repulsive potentials without physically meaningful terms for electrostatics, non-covalent interactions or atom solvation. To facilitate NMR structure calculations in the proper environment we are developing a computationally less demanding implicit solvent potential for the XPLOR-NIH NMR structure refinement package. Here we show that the potential provides significant improvements both in the quality and precision of the calculated structures; it improves the accuracy of structures determined with sparse restraints; it provides numerous physically meaningful hydrogen bonding connections; and it provides correct embedding of membrane proteins in lipid bilayer membranes.This research was supported by grants from the National Institutes of Health (AI074805; GM100265; GM094727; EB002031).

Minimal Viral Potassium Channels for Studying Protein/Lipid Interaction

The channel proteins from Chlorella viruses are miniature versions of K+ channels. They are the structural equivalents of the pore module of complex K+ channels from eukaryotes and they include the selectivity filter, the cavity and gates. The channel KcvNTS is with only 82 amino acids particularly small and molecular dynamics simulations suggest that the channel is quasi fully embedded in a di-myristoylphosphatidylcholin (C14) lipid bilayer [1]. This close interaction between the channel protein and its lipid environment offers the possibility to examine lipid/protein interaction for a channel pore in a systematic manner. For this purpose we reconstituted the purified channel protein in lipid bilayers with different chain lengths, phospholipid head groups and in the absence and presence of detergents or cholesterol. The analysis of single channel activity shows that the membrane composition affects channel gating but not the unitary conductance. Most interesting is a voltage dependency of the channel, which is introduced by the presence of cholesterol, detergent or bilayers with long chain length.Reference1. Braun, C.J., et al., Viral potassium channels as a robust model system for studies of membrane-protein interaction. Biochim Biophys Acta, 2013.

Paramagnetic doping of a 7TM membrane protein in lipid bilayers by Gd3+-complexes for solid-state NMR spectroscopy

A considerable limitation of NMR spectroscopy is its inherent low sensitivity. Approximately 90 % of the measuring time is used by the spin system to return to its Boltzmann equilibrium after excitation, which is determined by (1)H-T1 in cross-polarized solid-state NMR experiments. It has been shown that sample doping by paramagnetic relaxation agents such as Cu(2+)-EDTA accelerates this process considerably resulting in enhanced sensitivity. Here, we extend this concept to Gd(3+)-complexes. Their effect on (1)H-T1 has been assessed on the membrane protein proteorhodopsin, a 7TM light-driven proton pump. A comparison between Gd(3+)-DOTA, Gd(3+)-TTAHA, covalently attached Cu(2+)-EDTA-tags and Cu(2+)-EDTA reveals a 3.2-, 2.6-, 2.4- and 2-fold improved signal-to-noise ratio per unit time due to longitudinal paramagnetic relaxation enhancement. Furthermore, Gd(3+)-DOTA shows a remarkably high relaxivity, which is 77-times higher than that of Cu(2+)-EDTA. Therefore, an order of magnitude lower dopant concentration can be used. In addition, no line-broadening effects or peak shifts have been observed on proteorhodopsin in the presence of Gd(3+)-DOTA. These favourable properties make it very useful for solid-state NMR experiments on membrane proteins.

Nano-Chemical Infrared Imaging of Membrane Proteins in Lipid Bilayers

The spectroscopic characterization of biomolecular structures requires nanometer spatial resolution and chemical specificity. We perform full spatio-spectral imaging of dried purple membrane patches purified from Halobacterium salinarum with infrared vibrational scattering-type scanning near-field optical microscopy (s-SNOM). Using near-field spectral phase contrast based on the Amide I resonance of the protein backbone, we identify the protein distribution with 20 nm spatial resolution and few-protein sensitivity. This demonstrates the general applicability of s-SNOM vibrational nanospectroscopy, with potential extension to a wide range of biomolecular systems.

Lipid bilayer arrays: Cyclically formed and measured

Artificial lipid bilayers have many uses. They are well established for scientific studies of reconstituted ion channels, used to host engineered pore proteins for sensing, and can potentially be applied in DNA sequencing. Droplet bilayers have significant technological potential for enabling many of these applications due to their compatibility with automation and array platforms. To further develop this potential, we have simplified the formation and electrical measurement of droplet bilayers using an apparatus that only requires fluid dispensation. We achieved simultaneous bilayer formation and measurement over a 32-element array with ~80% yield and no operator input following fluid addition. Cycling these arrays resulted in the formation and measurement of 96 out of 120 possible bilayers in 80 minutes, a sustainable rate that could significantly increase with automation and greater parallelization. This turn-key, high-yield approach to making artificial lipid bilayers requires no training, making the capability of creating and measuring lipid bilayers and ion channels accessible to a much wider audience. In addition, this approach is low-cost, parallelizable, and automatable, allowing high-throughput studies of ion channels and pore proteins in lipid bilayers for sensing or screening applications.

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.

Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy.

The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD ~0.44 Å, a tilt angle of 24° ± 1°, and an azimuthal angle of 55° ± 6°. This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional oriented solid-state NMR and magic angle spinning solid-state NMR.