10 research outputs found
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-NH2) bind to CB7 with moderately high affinity (K ≈ 104 M–1), while their cleavage products (e.g., Phe-Met-NH2) bind very tightly (K \u3e 106 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 (−CONH2 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
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.</p
Direct observation of proton pumping by a eukaryotic P-type ATPase
In eukaryotes, P-type ATPases generate the plasma membrane potential and drive secondary transport systems; however, despite their importance, their regulation remains poorly understood. Here 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 non-equilibrium model of vesicle acidification, revealed that pumping is stochastically interrupted by long-lived (~100 s) 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