Designed membrane channels and pores

Advances in the synthesis and assembly of designed membrane channels and pores include addressable template-assisted synthetic protein (TASP) syntheses of helix bundles, the production of a new class of nanotubes and the ability to purify hetero-oligomeric pores. Channels and pores with altered functional properties and with built-in triggers and switches have been prepared. Progress in applications has been greatest in sensor technology, where sensor elements based on ligand activation, channel selectivity and channel block have been made. Structural information about natural membrane proteins is emerging to inspire new designs.     

Recent advances on proteins of plant terminal membranes.

Since the beginning of the 1990s, our knowledge of the protein equipment of plant membranes progresses at an accelerating pace, owing to the irruption of molecular biology tools and genetics strategies in plant biology. Map-based cloning strategies and exploration of EST databases rapidly enrich the catalog of cDNA or gene sequences expected to code for membrane proteins. The accumulation of 'putative' membrane proteins reinforces the need for structural, functional and physiological information. Indeed, ambiguities often exist concerning the association to a membrane, the membrane identity and the topology of the protein inserted in the membrane. The combination of directed mutagenesis and heterologous expression of plant genes in various systems and plant reverse genetics has opened the possibility to study molecular and physiological functions. This review will emphasize how these tools have been essential for the exciting recent discoveries on plant terminal membrane proteins. These discoveries concern a variety of transport systems for ions, organic solutes including auxin, water channels, a large collection of systems suspected to act as receptors of chemical signals, proteins thought to control vesicle trafficking and enzymatic systems.     

Optimization of bacteriorhodopsin for bioelectronic devices

Bacteriorhodopsin (BR) is the photoactive proton pump found in the purple membrane of the salt marsh archaeon Halobacterium salinarum. Evolution has optimized this protein for high photochemical efficiency, thermal stability and cyclicity, as the organism must be able to function in a hot, stagnant and resource-limited environment. Photonic materials generated via organic chemistry have yet to surpass the native protein in terms of quantum efficiency or cyclicity. However, the native protein still lacks the overall efficiency necessary for commercial viability and virtually all successful photonic devices using bacteriorhodopsin are based on chemical or genetic variants of the native protein. We show that genetic engineering can provide significant improvement in the device capabilities of proteins and, in the case of bacteriorhodopsin, a 700-fold improvement has been realized in volumetric data storage. We conclude that semi-random mutagenesis and directed evolution will play a prominent role in future efforts in bioelectronic optimization.     

Biomimetic design of nanopatterned membranes

The biomimetic approach copying the supramolecular building principle of many archaeal cell envelopes (i.e., a plasma membrane with associated S-layer proteins) has resulted in stable lipid membranes with excellent reconstitution properties for transmembrane proteins. This is a particular challenge as one-third of all proteins in an organism are membrane proteins like pores, ion channels, or receptors. At S-layer supported lipid membranes, spatial well-defined domains on the S-layer protein interact noncovalently with lipid head groups within the lipid membrane resulting in a nanopatterning of a few anchored and scores of diffusional free-lipid molecules. In addition, no impact on the hydrophobic core region and on the function of reconstituted integral proteins has been determined. Among others, particularly S-layer stabilized membranes can be used for structure-function studies on reconstituted integral proteins and also in the membrane protein-based molecular nanotechnology, e.g., in the design of biosensing devices (e.g., lipid chip or lab-on-a-chip), or for receptor or ion channel-based high-throughput screening.     

bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.     

Molecular packing and packing defects in helical membrane proteins

The packing of helices spanning lipid bilayers is crucial for the stability and function of alpha-helical membrane proteins. Using a modified Voronoi procedure, we calculated packing densities for helix-helix contacts in membrane spanning domains. Our results show that the transmembrane helices of protein channels and transporters are significantly more loosely packed compared with helices in globular proteins. The observed packing deficiencies of these membrane proteins are also reflected by a higher amount of cavities at functionally important sites. The cavities positioned along the gated pores of membrane channels and transporters are noticeably lined by polar amino acids that should be exposed to the aqueous medium when the protein is in the open state. In contrast, nonpolar amino acids surround the cavities in those protein regions where large rearrangements are supposed to take place, as near the hinge regions of transporters or at restriction sites of protein channels. We presume that the observed deficiencies of helix-helix packing are essential for the helical mobility that sustains the function of many membrane protein channels and transporters.     

Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

Amino acid residue arginine-158 of the outer membrane protein PhoE of Escherichia coli K-12 has been shown to be cell-surface-exposed [Korteland et al. (1985) Eur. J. Biochem. 152, 691-697]. To study the effects of small insertions in this region of the protein on its biogenesis and characteristics, a unique restriction site was created by site-directed mutagenesis in a plasmid carrying the phoE gene and oligonucleotides of 12-74 bp were inserted. The insertions did not interfere with incorporation into the outer membrane since (a) several monoclonal antibodies, directed against the cell-surface-exposed part of PhoE protein, bound to whole cells producing the altered proteins and (b) the proteins formed functional pores for the uptake of beta-lactam antibiotics. The binding of one monoclonal antibody and of the PhoE-specific phages TC45 and TC45hrN3 was disturbed by the insertions, showing that this region of the protein is immunogenic and is involved in the binding of both of these phages. The functioning of the mutant pores was characterized both in vivo by studying the uptake of beta-lactam antibiotics and in vitro after the reconstitution of the proteins in black lipid films. The pore characteristics changed depending on the nature of the inserted amino acids. Addition of a negatively charged amino acid resulted in decreased anion-selectivity, whereas insertion of a positive charge and deletion of a negative charge had only a small influence.     

Signal peptides open protein-conducting channels in E. coli.

Plasma membrane vesicles and protoplasts of Escherichia coli were fused to planar lipid bilayers and studied with electrophysiological techniques. Large transmembrane aqueous channels were opened when 0.2 nM LamB signal peptide was added to the cytoplasmic side of the membrane. These aqueous pores are similar in conductance to those previously observed in mammalian endoplasmic reticulum when puromycin is used to release and thus unplug nascent translocating chains. Signal sequences have been previously shown to be necessary and sufficient for targeting proteins to cellular membranes. These results demonstrate that signal peptides are sufficient for opening the protein-conducting channels. We suggest that they are the physiological ligands that open protein-conducting channels at the initiation of protein translocation across prokaryotic plasma membrane and mammalian endoplasmic reticulum.     

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

Summary Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15m KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.     

Structure and dynamics of the colicin E1 channel

The toxin-like and bactericidal colicin E1 molecule is of interest for problems of toxin action, polypeptide translocation across membranes, voltage-gated channels, and receptor function. Colicin E1 binds to a receptor in the outer membrane and is translocated across the cell envelope to the inner membrane. Import of the colicin channel-forming domain into the inner membrane involves a translocation-competent intermediate state and a membrane potential-dependent movement of one third to one half of the channel peptide into the membrane bilayer. The voltage-gated channel has a conductance sufficiently large to depolarize the Escherichia coli cytoplasmic membrane. Amino acid residues that affect the channel ion selectivity have been identified by site-directed mutagenesis. The colicin E1 channel is one of a few membrane proteins whose secondary structures in the membrane, predominantly alpha-helix, have been determined by physico-chemical techniques. Hypothesis for the identity of the trans-membrane helices, and the mechanism of binding to the membrane, are influenced by the solved crystal structure of the soluble colicin A channel peptide. The protective action of immunity protein is a unique aspect of the colicin problem, and information has been obtained, by genetic techniques, about the probable membrane topography of the imm gene product.     

Functional discrimination of membrane proteins using machine learning techniques

Background  

Discriminating membrane proteins based on their functions is an important task in genome annotation. In this work, we have analyzed the characteristic features of amino acid residues in membrane proteins that perform major functions, such as channels/pores, electrochemical potential-driven transporters and primary active transporters.     

A modification of the split-tobacco etch virus method for monitoring interactions between membrane proteins in mammalian cells

Despite progress in the development of methods to monitor protein interactions, studies of interactions between membrane proteins in mammalian cells remain challenging. Protein complementation assays (PCAs) are commonly used to study interactions between proteins due to their simplicity. They are based on interaction-mediated reconstitution of a reporter protein, which can be easily monitored. Recently, a protein complementation method named split-TEV (tobacco etch virus) has been developed and is based on the functional reconstitution of TEV protease and subsequent proteolytic-mediated activation of reporters. In this work, we have developed a modification of the split-TEV method to study the interactions between membrane proteins with increased specificity. This assay was validated by addressing the interactions between different membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels. By comparing it with another PCA, we found that this new method showed a higher sensitivity.     

Protein Structure Prediction and Design in a Biologically Realistic Implicit Membrane

Protein design is a powerful tool for elucidating mechanisms of function and engineering new therapeutics and nanotechnologies. Although soluble protein design has advanced, membrane protein design remains challenging because of difficulties in modeling the lipid bilayer. In this work, we developed an implicit approach that captures the anisotropic structure, shape of water-filled pores, and nanoscale dimensions of membranes with different lipid compositions. The model improves performance in computational benchmarks against experimental targets, including prediction of protein orientations in the bilayer, ΔΔG calculations, native structure discrimination, and native sequence recovery. When applied to de novo protein design, this approach designs sequences with an amino acid distribution near the native amino acid distribution in membrane proteins, overcoming a critical flaw in previous membrane models that were prone to generating leucine-rich designs. Furthermore, the proteins designed in the new membrane model exhibit native-like features including interfacial aromatic side chains, hydrophobic lengths compatible with bilayer thickness, and polar pores. Our method advances high-resolution membrane protein structure prediction and design toward tackling key biological questions and engineering challenges.     

微生物酶分子改造研究进展

近年来,越来越多的酶蛋白已经采用重组微生物反应器进行高效生产。为了改善酶蛋白的催化性能,提高其环境适应性,同时提高酶蛋白的表达量,降低生产成本,各种针对酶蛋白分子改造的基因工程技术已经得到大量的应用。综述了用于酶分子改造和进化的主要分子生物学方法,如定点突变、易错PCR、基因改组、密码子优化等技术及其应用成就。     

Mechanosensitive channels in bacteria: signs of closure?

Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer of the cytoplasmic membrane, where they transiently create large pores in a controlled manner. Mechanosensitive channel research has benefited from advances in electrophysiology, genomics and molecular genetics as well as from the application of biophysical techniques. Most recently, new analytical methods have been used to complement existing knowledge and generate insights into the molecular interactions that take place between mechanosensitive channel proteins and the surrounding membrane lipids. This article reviews the latest developments.     

Effect of solution pH on protein transport through ultrafiltration membranes

Although a number of previous studies have demonstrated that solution pH can have a dramatic effect on protein transport through ultrafiltration membranes, the exact origin of this behavior has been unclear. Experimental data were obtained for the transport of a broad range of proteins with different surface charge and molecular weight. The effective hydrodynamic size of the proteins was evaluated using size‐exclusion chromatography. The membrane charge, both before and after exposure to a given protein, was evaluated using streaming potential measurements. In most cases, the electrostatic interactions were dominated by the distortion of the electrical double layer surrounding the protein, leading to a distinct maximum in protein transmission at the protein isoelectric point. Attractive electrostatic interactions did occur when the protein and membrane had a large opposite charge, causing a second maximum in transmission at a pH between the isoelectric points of the protein and membrane. The sieving data were in good agreement with theoretical calculations based on available models for the partitioning of charged solutes in cylindrical pores. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 27–37, 1999.     

A GTP-binding protein activates chloride channels in a renal epithelium

Although G proteins have been shown to regulate cation channels, regulation of Cl- channels by G proteins has not been demonstrated directly. Accordingly, the objective of this study was to examine whether a G protein regulates Cl- channels in the apical membrane of rabbit kidney CCD cells grown in culture. Previous studies showed that this channel is activated by adenosine and protein kinase C and has a single channel conductance of 305 picosiemens. The PCl-:PNa+ is 9:1 and the PCl-:PHCO3- is 2:1 (Schwiebert, E.M., Light, D.B., Dietl, P., Fejes-Toth, G., Naray-Fejes-Toth, A., and Stanton, B. (1990) Kidney Int. 37,216). In the present study, Cl- channels in the apical membrane of CCD cells were studied by the patch clamp technique. GTP and guanosine 5'-O(3-thiophosphate) (GTP gamma S), a nonhydrolyzable analog of GTP, increased the single channel open probability (Po). In contrast, guanosine 5'-O-(2-thiophosphate), a nonhydrolyzable analog of GDP, and pertussis toxin (PTX) decreased the Po. GTP gamma S, but not GTP, reversed PTX inhibition of the channel. The alpha i-3-subunit of Gi increased the Po in both untreated and PTX-treated membrane patches. Because GTP gamma S activated the Cl- channel in the presence of H8, a protein kinase inhibitor, we conclude that the G protein does not activate the channel by stimulating a protein kinase. Thus, a PTX-sensitive G protein activates a Cl- channel in the apical membrane of renal CCD cells.     

Designing proteins to crystallize through beta-strand pairing

Inherent difficulties in growing protein crystals are major concerns within structural biology and particularly in structural proteomics. Here, we describe a novel approach of engineering target proteins by surface mutagenesis to increase the odds of crystallizing the molecules. To this end, we have exploited our recent triad-hypothesis using proteins with crystallographically defined beta-structures as the principal models. Crystal packing analyses of 182 protein structures belonging to 21 different superfamilies implied that the propensities to crystallize could be engineered into target proteins by replacing short segments, 5-6 residues, of their beta-strands with 'cassettes' of suitable packing motifs. These packing motifs will generate specific crystal packing interactions that promote crystallization. Key features of the primary and tertiary structures of such packing motifs have been identified for immunoglobulins. Further, packing motifs have been engineered successfully into six model antibodies without disturbing their capabilities to be produced, their immunoreactivity and their overall structure. Preliminary crystallization analyses have also been performed. Taken together, the procedures outline a rational protocol for crystallizing proteins by surface mutagenesis. The importance of these findings is discussed in relation to the crystallization of proteins in general.     

Fluorinated amphiphiles control the insertion of α-hemolysin pores into lipid bilayers

The insertion of fully folded and assembled ion channels and pores into planar lipid bilayers for electrical recording has been facilitated by the use of conventional detergents at a final concentration below the critical micelle concentration (CMC). After the desired number of channels or pores (often one) has been incorporated into a bilayer, it is important to prevent further insertion events, which is often done by awkward techniques such as perfusion. Here, we show that the addition of single-chain fluorinated amphiphiles (F-amphiphiles) with zwitterionic, simple neutral, and neutral oligomeric headgroups at a concentration above the CMC prevents the further insertion of staphylococcal α-hemolysin pores, MspA pores, and Kcv potassium channels into lipid bilayers. We found the commercially available F(6)FC (fluorinated fos-choline with a C(6)F(13)C(2)H(4) chain) to be the least perturbing and most effective agent for this purpose. Bilayers are known to be resistant to F-amphiphiles, which in this case we suppose sequester the pores and channels within amphiphile aggregates. We suggest that F-amphiphiles might be useful in the fabrication of bilayer arrays for nanopore sensor devices and the rapid screening of membrane proteins.