Landscape phage, phage display, stripped phage, biosensors, detection, affinity reagent, nanotechnology, Salmonella typhimurium, Bacillus anthracis

Filamentous phage, such as fd used in this study, are thread-shaped bacterial viruses. Their outer coat is a tube formed by thousands equal copies of the major coat protein pVIII. We constructed libraries of random peptides fused to all pVIII domains and selected phages that act as probes specific for a panel of test antigens and biological threat agents. Because the viral carrier is infective, phage borne bio-selective probes can be cloned individually and propagated indefinitely without needs of their chemical synthesis or reconstructing. We demonstrated the feasibility of using landscape phages and their stripped fusion proteins as new bioselective materials that combine unique characteristics of affinity reagents and self assembling membrane proteins. Biorecognition layers fabricated from phage-derived probes bind biological agents and generate detectable signals. The performance of phage-derived materials as biorecognition films was illustrated by detection of streptavidin-coated beads, Bacillus anthracis spores and Salmonella typhimurium cells. With further refinement, the phage-derived analytical platforms for detecting and monitoring of numerous threat agents may be developed, since the biodetector films may be obtained from landscape phages selected against any bacteria, virus or toxin. As elements of field-use detectors, they are superior to antibodies, since they are inexpensive, highly specific and strong binders, resistant to high temperatures and environmental stresses.Comment: Submitted on behalf of TIMA Editions (http://irevues.inist.fr/tima-editions

Alamethicin adsorption to a planar lipid bilayer.

The effect of alamethicin and its derivatives on the voltage-dependent capacitance of phosphatidylethanolamine (squalane) membranes was measured using two different methods: lock-in detection and voltage pulse. Alamethicin and its derivatives modulate the voltage-dependent capacitance at voltages lower than the voltage at which alamethicin-induced conductance is detected. The magnitude and sign of this alamethicin-induced capacitance change depends on the aqueous alamethicin concentration and the kind of alamethicin used. Our experimental data can be interpreted as a potential-dependent pseudocapacitance associated with adsorbed alamethicin. Pseudocapacitance is expressed as a function of alamethicin charge, its concentration in the bathing solution and the applied electric field. The theory describes the dependence of the capacitance on applied voltage and alamethicin concentration. When alamethicin is neutral the theory predicts no change of the voltage-dependent capacitance with either sign of applied voltage. Experimental data are consistent with the model in which alamethicin molecules interact with each other while being adsorbed to the membrane surface. The energy of this interaction depends on the alamethicin concentration

Thickness dependence of monoglyceride bilayer membrane conductance.

We have studied the conductance properties of unmodified monoglyceride membranes as a function of monoglyceride chain length. As membrane thickness decreases from 31 to 20 nm, the steepness of the current-voltage (I-V) curve increases from 80 mV per e-fold current increase to 52 mV per e-fold current increase. The zero-voltage conductance increases more than 1,000-fold and the apparent activation energy of conductance decreases from 18.4 to 14.2 kcal/mol. We have analyzed our results using both the Nernst-Planck equation and absolute rate theory. Both approaches are consistent with our results and give consistent values for the parameters describing the I-V curves. We conclude that both the surface ion concentration and the distance from the surface of the membrane at which the energy of an ion rises appreciably above its value in solution (position of the barrier) are invariant with thickness

Signal transduction across alamethicin ion channels in the presence of noise.

We have studied voltage-dependent ion channels of alamethicin reconstituted into an artificial planar lipid bilayer membrane from the point of view of electric signal transduction. Signal transduction properties of these channels are highly sensitive to the external electric noise. Specifically, addition of bandwidth-restricted "white" noise of 10-20 mV (r.m.s.) to a small sine wave input signal increases the output signal by approximately 20-40 dB conserving, and even slightly increasing, the signal-to-noise ratio at the system output. We have developed a small-signal adiabatic theory of stochastic resonance for a threshold-free system of voltage-dependent ion channels. This theory describes our main experimental findings giving good qualitative understanding of the underlying mechanism. It predicts the right value of the output signal-to-noise ratio and provides a reliable estimate for the noise intensity corresponding to its maximum. Our results suggest that the alamethicin channel in a lipid bilayer is a good model system for studies of mechanisms of primary electrical signal processing in biology showing an important feature of signal transduction improvement by a fluctuating environment

Probing alamethicin channels with water-soluble polymers. Effect on conductance of channel states.

Channel access resistance has been measured to estimate the characteristic size of a single ion channel. We compare channel conductance in the presence of nonpenetrating water-soluble polymers with that obtained for polymer-free electrolyte solution. The contribution of the access resistance to the total alamethicin channel resistance is approximately 10% for first three open channel levels. The open alamethicin channel radii inferred for these first three levels from the access resistance are 6.3, 10.3, and 11.4 A. The dependence of channel conductance on polymer molecular weight also allows evaluation of the channel dimensions from polymer exclusion. Despite varying conductance, it was shown that steric radii of the alamethicin channel at different conductance levels remain approximately unchanged. These results support a model of the alamethicin channel as an array of closely packed parallel pores of nearly uniform diameter