- Open Access
Nanopores: maltoporin channel as a sensor for maltodextrin and lambda-phage
© Berkane et al; licensee BioMed Central Ltd. 2005
- Received: 18 September 2004
- Accepted: 02 March 2005
- Published: 02 March 2005
To harvest nutrition from the outside bacteria e.g. E. coli developed in the outer cell wall a number of sophisticated channels called porins. One of them, maltoporin, is a passive specific channel for the maltodextrin uptake. This channel was also named LamB as the bacterial virus phage Lambda mis-uses this channel to recognise the bacteria. The first step is a reversible binding followed after a lag phase by DNA injection. To date little is known about the binding capacity and less on the DNA injection mechanism. To elucidate the mechanism and to show the sensitivity of our method we reconstituted maltoporin in planar lipid membranes. Application of an external transmembrane electric field causes an ion current across the channel. Maltoporin channel diameter is around a few Angstroem. At this size the ion current is extremely sensitive to any modification of the channels surface. Protein conformational changes, substrate binding etc will cause fluctuations reflecting the molecular interactions with the channel wall. The recent improvement in ion current fluctuation analysis allows now studying the interaction of solutes with the channel on a single molecular level.
We could demonstrate the asymmetry of the bacterial phage Lambda binding to its natural receptor maltoporin.
We suggest that this type of measurement can be used as a new type of biosensors.
- Single molecule detection
- Nanopore concept
Nature created and optimized proteins for specific tasks which makes them often interesting in material science. For example, membrane transporters could control the permeability of artificial nanometer sized container. A typical application could be to control the enzymatic activity in a liposome . Another possible application is to reconstitute channels into planar lipid bilayer and use time dependent conductance as a signal [2, 3]. Application of an external electric field drives the ions through the nano (and subnano) meter sized channel. Any larger molecule that diffuses into and temporarily sticks to the channel interior will cause typical fluctuations of the ion current which allow to conclude on its mode of translocation. Such studies were used to follow sugar translocation through maltoporin . Similar types of measurements were done to investigate the translocation of antibiotics like ampicillin . Subtle changes in the channel size or small conformational changes can be recorded and this technique could be developed towards an instrument to probe very soft forces.
Porins are attractive candidates for applications because they are very stable. Moreover, recombinant technology permits production of porins in E. coli with high yields . A third advantage is the availability of the high resolution 3-D crystal structure showing details of substrate binding sites which facilitates enormously a rational engineering of modified proteins.
The outer cell wall of Gram-negative bacteria from E. coli is fairly permeable to smaller solutes below a molecular weight of about 400 Da . Such substances can freely permeate under a concentration gradient through general diffusion porins in the outer cell wall. Under stress, e.g. in case of lack of nutrition, the pure diffusion process is too slow and the bacteria need to improve the efficiency of the translocation. For those cases, nature has created a series of rather specific and highly sophisticated membrane channels. The most extensively studied examples of specific porins are the maltooligosaccharide-specific channel Maltoporin of E. coli [4, 7, 8]. Maltoporin forms ion-conducting channels when reconstituted into lipid bilayers [9, 10]. The 3D structure of Maltoporin revealed that the monomer of Maltoporin of E. coli consists of an 18 stranded β-barrel with short turns at the periplasmic side and large irregular loops at the outside of the cell .
The bacteriophage Lambda is a virus recognizing Maltoporin at the outer cell surface . In absence of this membrane channel, phage Lambda does not recognize the bacteria. Or, even minor mutations allow the bacteria to defend themselves against virus attacks. The virus itself can, in turn mutate to restore binding ability. According to the high resolution X-ray structure the water filled channel is far too small to permit the translocation of the double strain DNA (about 20 Å) . The infection mechanism thus must involve one of the following processes: Phage binding will cause a strong conformational change within the Maltoporin or, after binding the phage releases a DNA translocation machinery to bring its DNA across the hydrophobic membrane. To date none of these intermediate steps has been observed so far and the underlying process remains unclear. Recently, gpJ, a protein in the phage terminal was identified to be involved in the Maltoporin recognition process .
Membrane current was measured via homemade Ag/AgCl electrodes. One electrode was used as ground and the other connected to the headstage of an Axopatch 200B amplifier (Axon Instruments, USA), allowing the application of adjustable potentials (typically, 100 mV) across the membrane. A similar set-up was used in the second measurement.
Sensing with membrane channel is a new way in screening for solute molecules and several promising examples are already shown [2, 3, 16, 19, 20]. The actual bottleneck is the complexity in membrane channel assembly. However, the current development in automatized patch-clamping will open a wide range of possibilities [21, 22]. We plan to reduce the volume on each side of the membrane and the size of the lipid patch. We currently work with pore diameters of about 1 μm with less background capacitance and thus a better time resolution and to simplify the channel assembly.
- Colletier J-P, Chaize B, Winterhalter M, Fournier D: Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer. BMC Biotechnology. 2002, 2: 9-17. 10.1186/1472-6750-2-9.View ArticleGoogle Scholar
- Howorka S, Nam J, Bayley H, Kahne D: Stochastic detection of monovalent and bivalent protein-ligand interactions. Angew Chemie Int Ed. 2004, 43: 842-846. 10.1002/anie.200352614.View ArticleGoogle Scholar
- Bezrukov SM: Ion channels as molecular coulter counters to probe metabolite transport. J Membr Biol. 2000, 174: 1-13. 10.1007/s002320001026.View ArticleGoogle Scholar
- Kullman L, Winterhalter M, Bezrukov SM: Transport of maltodextrins through maltoporin: A single-channel study. Biophys J. 2002, 82: 803-812.View ArticleGoogle Scholar
- Nestorovich EM, Danelon C, Winterhalter M, Bezrukov SM: Designed to penetrate: Time-resolved interaction of single antibiotic molecules with bacterial pores. Proc Natl Acad Sci (USA). 2002, 99: 9789-94. 10.1073/pnas.152206799.View ArticleGoogle Scholar
- Van Gelder P, Dumas F, Winterhalter M: Understanding the function of bacterial outer membrane channels by reconstitution into black lipid membranes. Biophys Chem. 2000, 85: 153-67. 10.1016/S0301-4622(99)00153-2.View ArticleGoogle Scholar
- Szmelcman S, Hofnung M: Maltose transport in Escherichia coli K12. Involvement of the bacteriophage lambda receptor. J Bacteriol. 1975, 124: 112-118.Google Scholar
- Schmid K, Ebner R, Altenbuchner J, Schmitt R, Lengeler JW: Plasmid-mediated sucrose metabolism in Escherichia coli K12: mapping of the scr genes of pUR400. Mol Microbiol. 1988, 2: 1-8.View ArticleGoogle Scholar
- Szmelcman S, Schwartz M, Silhavy TJ, Boos W: Maltose transport in Escherichia coli K-12: a comparison of transport kinetics in wild-type and lambda-resistant mutants with the dissociation constant of the maltose-binding protein as measured by fluorescent quenching. Eur J Biochem. 1976, 65: 13-19. 10.1111/j.1432-1033.1976.tb10383.x.View ArticleGoogle Scholar
- Benz R, Schmid A, Nakae T, Vos-Scheperkeuter GH: Pore formation by LamB of Escherichia coli in lipid bilayer membranes. J Bacteriol. 1986, 165: 978-986.Google Scholar
- Dutzler R, Wang Y-F, Rizkallah PJ, Rosenbusch JP, Schirmer T: Crystal structures of various maltooligosaccharides bound to Maltoporin reveal a specific sugar translocation pathway. Structure. 1996, 4: 127-134. 10.1016/S0969-2126(96)00016-0.View ArticleGoogle Scholar
- Roa M, Scandella D: Multiple steps during the interaction between coliphage lambda and its receptor protein in vitro. Virology. 1976, 72: 182-194. 10.1016/0042-6822(76)90322-6.View ArticleGoogle Scholar
- Wang J, Hofnung M, Charbit A: The C-terminal portion of the tail fiber protein of bacteriophage lambda is responsible for binding to LamB, its receptor at the surface of Escherichia coli K-12. J Bact. 2000, 182: 508-512. 10.1128/JB.182.2.508-512.2000.View ArticleGoogle Scholar
- Montal M, Mueller P: Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc Nat Acad Sci (USA). 1972, 69: 3561-3566.View ArticleGoogle Scholar
- Benz R, Janko K, Boos W, Läuger P: Formation of large, ion permeable membrane channels by matrix protein (porin) of Escherichia coli. Biochem Biophys Acta. 1978, 511: 305-319.View ArticleGoogle Scholar
- Danelon C, Brando T, Winterhalter M: Probing the orientation of reconstituted Maltoporin channels at the single-protein level. J Biol Chem. 2003, 278: 35542-51. 10.1074/jbc.M305434200.View ArticleGoogle Scholar
- Van Gelder P, Dumas F, Rosenbusch J, Winterhalter M: Oriented channels reveal asymmetric energy barriers for sugar translocation through maltoporin of Escherichia coli. Eur J Biochem. 2000, 267: 79-84. 10.1046/j.1432-1327.2000.00960.x.View ArticleGoogle Scholar
- Andersen C, Schiffler B, Charbit A, Benz R: PH-induced collapse of the extracellular loops closes Escherichia coli maltoporin and allows the study of asymmetric sugar binding. J Biol Chem. 2002, 277: 41318-25. 10.1074/jbc.M206804200.View ArticleGoogle Scholar
- Akeson M, Branton D, Kasianowicz JJ, Brandin E, Deamer DW: Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys J. 1999, 77: 3227-33.View ArticleGoogle Scholar
- Kasianowicz JJ, Burden DL, Han LC, Cheley S, Bayley H: Genetically engineered metal ion binding sites on the outside of a channel's transmembrane beta-barrel. Biophys J. 1999, 76: 837-45.View ArticleGoogle Scholar
- Bruggemann A, George M, Klau M, Beckler M, Steindl J, Behrends JC, Fertig N: High quality ion channel analysis on a chip with the NPC technology. Assay Drug Dev Technol. 2003, 1: 665-673. 10.1089/154065803770381020.View ArticleGoogle Scholar
- Schmidt C, Mayer M, Vogel H: A Chip-Based Biosensor for the Functional Analysis of Single Ion Channels. Angew Chem Int Ed Engl. 2000, 39: 3137-3140. 10.1002/1521-3773(20000901)39:17<3137::AID-ANIE3137>3.0.CO;2-D.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.