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Catalytic activity of a synthetic multifunctional pore is studied in large unilamellar vesicles under conditions where substrate and synthetic catalytic pore (SCP) approach the membrane either from the same side (cis catalysis) or from opposite sides (trans catalysis). A synthetic supramolecular rigid-rod beta-barrel with excellent ion channel characteristics is identified as SCP using 8-acetoxypyrene-1,3,6-trisulfonate (AcPTS) as model substrate. The key finding is that application of supportive membrane potentials increases the initial velocity of AcPTS esterolysis (v0). This results in an increase of Vmax beyond experimental error (+30%), whereas KM increases less significantly. Long-range electrostatic steering by the membrane potential, possibly guiding substrates into the transmembrane catalyst and, more importantly, accelerating product release (foff = 1.3) is discussed as one possible explanation of this global reduction of catalyst saturation. Control experiments show, inter alia, that similarly strong changes do not occur with opposing membrane potentials.

Recently, synthetic multifunctional pores have been identified as "universal" detectors of chemical reactions. In this report, we show that with the assistance of enzymes as variable co-sensors, synthetic multifunctional pores can serve as similar universal sensors of variable components in mixed analytes. Sugar sensing in soft drinks is used to exemplify this new concept. This is achieved using invertase and hexokinase as co-sensors and a new synthetic multifunctional pore capable of discriminating between ATP and ADP in an "on-off" manner as sensor. The on-off discrimination between ATP as good and ADP as poor pore blocker is shown to be reasonably tolerant of changing experimental conditions. These results identify universal sensing with synthetic multifunctional pores as a robust, sensitive, and noninvasive method with appreciable promise for practical applications.

We report the characterization of multifunctional rigid-rod β-barrel ion channels with either internal aspartates or arginine-histidine dyads by planar bilayer conductance experiments. Barrels with internal aspartates form cation selective, large, unstable and ohmic barrel-stave (rather than toroidal) pores; addition of magnesium cations nearly deletes cation selectivity and increases single-channel stability. Barrels with internal arginine-histidine dyads form cation selective (P K ϩ/P Cl Ϫ = 2.1), small and ohmic ion channels with superb stability (single-channel lifetime > 20 seconds). Addition of "protons" results in inversion of anion/cation selectivity (P Cl Ϫ/P K ϩ = 3.8); addition of an anionic guest (HPTS) results in the blockage of anion selective but not cation selective channels. These results suggest that specific, internal counterion immobilization, here magnesium (but not sodium or potassium) cations by internal aspartates and inorganic phosphates by internal arginines (but not histidines), provides access to synthetic multifunctional pores with attractive properties.

This report demonstrates that a single set of identical synthetic multifunctional pores can detect the activity of many different enzymes. Enzymes catalyzing either synthesis or degradation of DNA (exonuclease III or polymerase I), RNA (RNase A), polysaccharides (heparinase I, hyaluronidase, and galactosyltransferase), and proteins (papain, ficin, elastase, subtilisin, and pronase) are selected to exemplify this key characteristic of synthetic multifunctional pore sensors. Because anionic, cationic, and neutral substrates can gain access to the interior of complementarily functionalized pores, such pores can be the basis for very userfriendly screening of a broad range of enzymes. There are compelling reasons to believe that the ''universal enzyme sensor,'' a user-friendly, noninvasive device that can detect all existing enzyme activities, belongs to the world of fiction and, despite functional proteomics, will never become reality (1, 2, ‡). However, it would be erroneous to conclude that efforts to maximize adaptability of noninvasive enzyme sensors to as many enzymes as possible are a simple waste of time. In this study, selected examples from biopolymer enzymology are used to demonstrate that a set of identical synthetic multifunctional pores (SMPs) can be used for noninvasive detection of the activity of many different enzymes (Fig. 1).In brief, if substrates bind and block the pore better than products, enzyme activity gradually removes the blocking agent and the pore can open. On the other hand, if products bind and block the pores better than the substrates, enzyme activity gradually produces blocking agents that can block the pores (Fig. 2). Thus, if there is substantial molecular recognition of either substrate or product by the same pore, the only prerequisite for detecting enzyme activities with SMP sensors is a simple method to distinguish between blocked and unblocked pores.As SMPs we introduced rigid-rod ␤-barrels 1 and 2 ( Fig. 2C) (2). Whereas various other rigid-rod ␤-barrel SMPs are available to cope with particular sensing requirements, SMPs other than rigid-rod ␤-barrels remain underexplored (3-9), particularly when compared with the remarkable progress made with bioengineered multifunctional pores as stochastic sensors of single analytes (10-13).The syntheses of barrel-stave supramolecules 1 (14) and 2 (15) from commercial biphenyl and amino acid derivatives in 19 steps overall each have been described. The characteristics of pores formed by rigid-rod ␤-barrels 1 and 2 in spherical and planar lipid bilayer membranes have also been reported (2, 14-16). Even without optimization, the extraordinary permeabilizing activity of these p-octiphenyl ␤-barrels makes it possible to perform Ͼ300,000 enzyme assays per mg of polypeptide (1).L-histidine (H) and L-arginine (R) residues at the inner barrel surface account for the multifunctionality of pore 1. Lining the ion-conducting pathway of SMP 1, these cationic residues recognize anionic substrates and products Ͼ10,000 times better than biological po...

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This account summarizes five years of research devoted to the development of the concept of synthetic multifunctional pores. The objective is to complement a comprehensive graphical summary of molecular recognition with a survey of structural studies on the same topic. The relevance of the latter for research focusing on creation and application of supramolecular functional materials is discussed briefly in a subjective manner.

This report delineates scope and limitation of the selectivity of synthetic multifunctional pores as enzyme sensors using glycolytic enzymes as example (G. Das, P. Talukdar, and S. Matile, Science, 2002, Vol. 298, pp. 1600-1602). Unproblematic detectability of hexokinase and phosphofructokinase demonstrates that the selectivity of synthetic multifunctional pore (SMPs) sensors suffices to sense ATP in mixed analytes containing ADP, whereas detection of the isomerization of glucose 6-phosphate into fructose 6-phosphate by phosphoglucose isomerase is not possible with confidence. The sensitivity of SMP sensors is sufficient for end-point detection of one picomole poly-L-glutamate hydrolyzed by papain in unoptimized assay format; the sensitivity of melittin as representative biological pore of similar charge and aggregation number to detect the same reaction is more than four orders of magnitude inferior.