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

Fiber-MOPA sources of coherent radiation

This paper presents the concept and design of medium to high power fiber radiation sources in MOPA (Master Oscillator Power Amplifier) configuration. Research results are presented on a two-stage power amplifier for MOPA applications for the third telecommunications window at 1550 nm based on active erbium-doped and erbium/ytterbium-doped fibers. The amplifier is fabricated entirely in fiber technology and delivers several watts of output power

Experimental verification of the method for producing a three-dimensional cross-pairs metamaterial structure based on a dielectric AlN cube

This paper presents a method of manufacturing 3D metamaterial structures using laser radiation. Our proposed material, aluminum nitride (AlN), by interaction of a high-energy laser beam undergoes a direct metallization process, a result of which a conductive aluminum layer is formed on the ceramic surface at the spot of the laser radiation. Using this unique feature on all six sides of an AlN ceramic cube, the designed cross-pairs were mapped. Unit cells were exposed to an electromagnetic wave, by which the occurrence of the resonant areas of reflectance and transmittance for 7.27 GHz and 9.14 GHz was observed. To demonstrate the property of the negative refractive index a prism made out of manufactured cubes was created. For a wave propagating through a wedge-shaped prism for certain frequency bands, a negative refraction angle was observed

Probing eudesmane cation−π interactions in catalysis by aristolochene synthase with non-canonical amino acids

Stabilization of the reaction intermediate eudesmane cation (3) through interaction with Trp 334 during catalysis by aristolochene synthase from Penicillium roqueforti was investigated by site-directed incorporation of proteinogenic and non-canonical aromatic amino acids. The amount of germacrene A (2) generated by the mutant enzymes served as a measure of the stabilization of 3. 2 is a neutral intermediate, from which 3 is formed during PR-AS catalysis by protonation of the C6,C7 double bond. The replacement of Trp 334 with para-substituted phenylalanines of increasing electron-withdrawing properties led to a progressive accumulation of 2 that showed a good correlation with the interaction energies of simple cations such as Na+ with substituted benzenes. These results provide compelling evidence for the stabilizing role played by Trp 334 in aristolochene synthase catalysis for the energetically demanding transformation of 2 to 3