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.
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.
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