Fluorescent artificial receptor-based membrane assay (FARMA) for spatiotemporally resolved monitoring of biomembrane permeability.

The spatiotemporally resolved monitoring of membrane translocation, e.g., of drugs or toxins, has been a long-standing goal. Herein, we introduce the fluorescent artificial receptor-based membrane assay (FARMA), a facile, label-free method. With FARMA, the permeation of more than hundred organic compounds (drugs, toxins, pesticides, neurotransmitters, peptides, etc.) through vesicular phospholipid bilayer membranes has been monitored in real time (µs-h time scale) and with high sensitivity (nM-µM concentration), affording permeability coefficients across an exceptionally large range from 10-9-10-3 cm s-1. From a fundamental point of view, FARMA constitutes a powerful tool to assess structure-permeability relationships and to test biophysical models for membrane passage. From an applied perspective, FARMA can be extended to high-throughput screening by adaption of the microplate reader format, to spatial monitoring of membrane permeation by microscopy imaging, and to the compartmentalized monitoring of enzymatic activity.

Direct observation of proton pumping by a eukaryotic P-type ATPase.

In eukaryotes, P-type adenosine triphosphatases (ATPases) generate the plasma membrane potential and drive secondary transport systems; however, despite their importance, their regulation remains poorly understood. We monitored at the single-molecule level the activity of the prototypic proton-pumping P-type ATPase Arabidopsis thaliana isoform 2 (AHA2). Our measurements, combined with a physical nonequilibrium model of vesicle acidification, revealed that pumping is stochastically interrupted by long-lived (~100 seconds) inactive or leaky states. Allosteric regulation by pH gradients modulated the switch between these states but not the pumping or leakage rates. The autoinhibitory regulatory domain of AHA2 reduced the intrinsic pumping rates but increased the dwell time in the active pumping state. We anticipate that similar functional dynamics underlie the operation and regulation of many other active transporters.

Dynamically analyte-responsive macrocyclic host-fluorophore systems.

CONSPECTUS: Host-guest chemistry commenced to a large degree with the work of Pedersen, who in 1967 first reported the synthesis of crown ethers. The past 45 years have witnessed a substantial progress in the field, from the design of highly selective host molecules as receptors to their application in drug delivery and, particularly, analyte sensing. Much effort has been expended on designing receptors and signaling mechanism for detecting compounds of biological and environmental relevance. Traditionally, the design of a chemosensor comprises one component for molecular recognition, frequently macrocycles of the cyclodextrin, cucurbituril, cyclophane, or calixarene type. The second component, used for signaling, is typically an indicator dye which changes its photophysical properties, preferably its fluorescence, upon analyte binding. A variety of signal transduction mechanisms are available, of which displacement of the dye from the macrocyclic binding site is one of the simplest and most popular ones. This constitutes the working principle of indicator displacement assays. However, indicator displacement assays have been predominantly exploited in a static fashion, namely, to determine absolute analyte concentrations, or, by using combinations of several reporter pairs, to achieve a differential sensing and, thus, identification of specific food products or brands. In contrast, their use in biological systems, for example, with membranes, cells, or with enzymes has been comparably less explored, which led us to the design of the so-called tandem assays, that is, dynamically analyte-responsive host-dye systems, in which the change in analyte concentrations is induced by a biological reaction or process. This methodological variation has practical application potential, because the ability to monitor these biochemical pathways or to follow specific molecules in real time is of paramount interest for both biochemical laboratories and the pharmaceutical industry. We will begin by describing the underlying principles that govern the use of macrocycle-fluorescent dye complexes to monitor time-dependent changes in analyte concentrations. Suitable chemosensing ensembles are introduced, along with their fluorescence responses (switch-on or switch-off). This includes supramolecular tandem assays in their product- and substrate-selective variants, and in their domino and enzyme-coupled modifications, with assays for amino acid decarboxylases, diamine, and choline oxidase, proteases, methyl transferases, acetylcholineesterase (including an unpublished direct tandem assay), choline oxidase, and potato apyrase as examples. It also includes the very recently introduced tandem membrane assays in their published influx and unpublished efflux variants, with the outer membrane protein F as channel protein and protamine as bidirectionally translocated analyte. As proof-of-principle for environmental monitoring applications, we describe sensing ensembles for volatile hydrocarbons.

Chemosensing ensembles for monitoring biomembrane transport in real time.

The efficacy of drugs and biomolecules relies on their ability to pass through the bilayer. The development of methods to directly and sensitively monitor these membrane transport processes has remained an experimental challenge. A macrocyclic host (p-sulfonatocalix[4]arene or cucurbit[7]uril) and a fluorescent dye (lucigenin or berberine) are encapsulated as a chemosensing ensemble inside liposomes, which allows for a direct, real-time fluorescence monitoring of the passage of unlabeled bioorganic analytes. This in vitro assay is transferable to different channel proteins and analytes, has potential for fluorescence-based screening, e.g., of channel modulators, and yields the absolute kinetics of translocation. Using this new biophysical method, we observed for the first time direct rapid translocation of protamine, an antimicrobial peptide, through the bacterial transmembrane protein OmpF.

Monitoring stepwise proteolytic degradation of peptides by supramolecular domino tandem assays and mass spectrometry for trypsin and leucine aminopeptidase.

A label-free optical detection method has been designed that allows direct monitoring of enzymatic peptide digestion in vitro. The method is based on the addition of a reporter pair, composed of the macrocyclic host cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO), to detect the proteolytic degradation of peptides. The enzymatic activity of trypsin and leucine aminopeptidase (LAP) was investigated using H-LSRFSWGA-OH as a substrate. The substrate as well as the intermediary and final products (i.e., H-FSWGA-OH and phenylalanine) formed during its enzymatic hydrolysis differ in their binding affinity to the receptor CB7, which results in varying degrees of dye displacement and, therefore, different fluorescence intensities. CB7 showed a relatively weak binding constant of K approximately 10(4) M(-1) with the substrate, a relatively strong binding constant of K > or = 10(6) M(-1) with H-FSWGA-OH (which is a final product formed by trypsin digestion and the intermediary product formed during the enzymatic activity of LAP), and a moderate binding constant of K < or = 10(5) M(-1) with phenylalanine. Owing to this differential binding affinity of CB7 with the substrate and the corresponding products, the digestion of a peptide by trypsin was followed as a decrease in fluorescence signal, while the complete degradation of the peptide by LAP was monitored as a decrease and a subsequent increase in fluorescence signal. The k(cat)/K(M) value for trypsin (2.0 x 10(7) min(-1) M(-1)) was derived from the change in fluorescence signal with time. Additionally, the complete degradation of the peptide by LAP was also followed by mass spectrometry. The use of a supramolecular sensing ensemble (macrocyclic host and dye) as a fluorescent reporter pair gives this method the flexibility to adapt for monitoring the stepwise degradation of different biologically relevant peptides by other proteases.

Determining protease substrate selectivity and inhibition by label-free supramolecular tandem enzyme assays.

An analytical method has been developed for the continuous monitoring of protease activity on unlabeled peptides in real time by fluorescence spectroscopy. The assay is enabled by a reporter pair comprising the macrocycle cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO). CB7 functions by selectively recognizing N-terminal phenylalanine residues as they are produced during the enzymatic cleavage of enkephalin-type peptides by the metalloendopeptidase thermolysin. The substrate peptides (e.g., Thr-Gly-Ala-Phe-Met-NH(2)) bind to CB7 with moderately high affinity (K ≈ 10(4) M(-1)), while their cleavage products (e.g., Phe-Met-NH(2)) bind very tightly (K > 10(6) M(-1)). AO signals the reaction upon its selective displacement from the macrocycle by the high affinity product of proteolysis. The resulting supramolecular tandem enzyme assay effectively measures the kinetics of thermolysin, including the accurate determination of sequence specificity (Ser and Gly instead of Ala), stereospecificity (d-Ala instead of l-Ala), endo- versus exopeptidase activity (indicated by differences in absolute fluorescence response), and sensitivity to terminal charges (-CONH(2) vs -COOH). The capability of the tandem assay to measure protease inhibition constants was demonstrated on phosphoramidon as a known inhibitor to afford an inhibition constant of (17.8 ± 0.4) nM. This robust and label-free approach to the study of protease activity and inhibition should be transferable to other endo- and exopeptidases that afford products with N-terminal aromatic amino acids.

Substrate-selective supramolecular tandem assays: monitoring enzyme inhibition of arginase and diamine oxidase by fluorescent dye displacement from calixarene and cucurbituril macrocycles.

A combination of moderately selective host-guest binding with the impressive specificity of enzymatic transformations allows the real-time monitoring of enzymatic reactions in a homogeneous solution. The resulting enzyme assays ("supramolecular tandem assays") exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. Two examples of enzymatic reactions were investigated: the hydrolysis of arginine to ornithine catalyzed by arginase and the oxidation of cadaverine to 5-aminopentanal by diamine oxidase, in which the substrates have a higher affinity to the macrocycle than the products ("substrate-selective assays"). The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene was used as the macrocycle, which displayed binding constants of 6400 M(-1) with arginine, 550 M(-1) with ornithine, and 60,000 M(-1) with the selected fluorescent dye (1-aminomethyl-2,3-diazabicyclo[2.2.2]oct-2-ene); the dye shows a weaker fluorescence in its complexed state, which leads to a switch-off fluorescence response in the course of the enzymatic reaction. For diamine oxidase, cucurbit[7]uril (CB7) was used as the macrocycle, which showed binding constants of 4.5 x 10(6) M(-1) with cadaverine, 1.1 x 10(5) M(-1) with 1-aminopentane (as a model for the thermally unstable 1-aminopentanal), and 2.9 x 10(5) M(-1) with the selected fluorescent dye (acridine orange, AO); AO shows a stronger fluorescence in its complexed state, which leads to a switch-on fluorescence response upon enzymatic oxidation. It is demonstrated that tandem assays can be successfully used to probe the inhibition of enzymes. Inhibition constants were estimated for the addition of known inhibitors, i.e., S-(2-boronoethyl)-L-cysteine and 2(S)-amino-6-boronohexanoic acid for arginase and potassium cyanide for diamine oxidase. Through the sequential coupling of a "product-selective" with a "substrate-selective" assay it was furthermore possible to monitor a multistep biochemical pathway, namely the decarboxylation of lysine to cadaverine by lysine decarboxylase followed by the oxidation of cadaverine by diamine oxidase. This "domino tandem assay" was performed in the same solution with a single reporter pair (CB7/AO).

Effects of cucurbit[7]uril on enzymatic activity.

The macrocyclic host cucurbit[7]uril exhibits highly specific inhibitory effects on the activity of proteases, which can be analyzed by a host-substrate complexation model.