496 research outputs found
Stimuli-Responsive Polyguanidino-Oxanorbornene Membrane Transporters as Multicomponent Sensors in Complex Matrices
We introduce guanidinium-containing synthetic polymers based on polyguanidino-oxanorbornenes (PGONs) as anion transporters in lipid bilayers that can be activated and inactivated by chemical stimulation. According to fluorogenic anion export experiments with vesicles, PGON transporters are most active in neutral bilayers near their phase transition, with EC50's in the nanomolar range. Six times higher effective transporter concentrations were measured with aminonaphthalene-1,3,6-trisulfonate than with 5(6)-carboxyfluorescein, demonstrating the importance of anion binding for transport and excluding nonspecific efflux. Negative surface potentials efficiently annihilate transport activity, while inside-negative membrane potentials slightly increase it. These trends demonstrate the functional importance of counterions to hinder the binding of hydrophilic counterions and to minimize the global positive charge of the transporter-counterion complexes. Strong, nonlinear increases in activity with polymer length reveal a significant polymer effect. Overall, the characteristics of PGONs do not match those of similar systems (for example, polyarginine) and hint toward an interesting mode of action, clearly different from nonspecific leakage caused by detergents. The activity of PGONs increases in the presence of amphiphilic anions such as pyrenebutyrate (EC50 = 70 microM), while several other amphiphilic anions tested were inactive. PGONs are efficiently inactivated by numerous hydrophilic anions including ATP (IC 50 = 150 microM), ADP (IC50 = 460 microM), heparin (IC50 = 1.0 microM), phytate (IC50 = 0.4 microM), and CB hydrazide (IC50 = 26 microM). The compatibility of this broad responsiveness with multicomponent sensing in complex matrices is discussed and illustrated with lactate sensing in sour milk. The PGON lactate sensor operates together with lactate oxidase as a specific signal generator and CB hydrazide as an amplifier for covalent capture of the pyruvate product as CB hydrazone (IC50 = 1.5 microM)
AnionâÏ Enzymes
In this report, we introduce artificial enzymes that operate with anion-Ï interactions, an interaction that is essentially new to nature. The possibility to stabilize anionic intermediates and transition states on an Ï-acidic surface has been recently demonstrated, using the addition of malonate half thioesters to enolate acceptors as a biologically relevant example. The best chiral anion-Ï catalysts operate with an addition/decarboxylation ratio of 4:1, but without any stereoselectivity. To catalyze this important but intrinsically disfavored reaction stereoselectively, a series of anion-Ï catalysts was equipped with biotin and screened against a collection of streptavidin mutants. With the best hit, the S112Y mutant, the reaction occurred with 95% ee and complete suppression of the intrinsically favored side product from decarboxylation. This performance of anion-Ï enzymes rivals, if not exceeds, that of the best conventional organocatalysts. Inhibition of the S112Y mutant by nitrate but not by bulky anions supports that contributions from anion-Ï interactions exist and matter, also within proteins. In agreement with docking results, K121 is shown to be essential, presumably to lower the p K a of the tertiary amine catalyst to operate at the optimum pH around 3, that is below the p K a of the substrate. Most importantly, increasing enantioselectivity with different mutants always coincides with increasing rates and conversion, i.e., selective transition-state stabilization
Anion-Ï catalysis: Bicyclic products with four contiguous stereogenic centers from otherwise elusive diastereospecific domino reactions on Ï-acidic surfaces
AnionâÏ interactions have been introduced recently to catalysis. The idea of stabilizing anionic intermediates and transition states on Ï-acidic surfaces is a new fundamental concept. By now, examples exist for asymmetric enolate, enamine, iminium and transamination chemistry, and the first anionâÏ enzyme has been created. Delocalized over large aromatic planes, anionâÏ interactions appear particularly attractive to stabilize extensive long-distance charge displacements during domino processes. Moving on from the formation of cyclohexane rings with five stereogenic centers in one step on a Ï-acidic surface, we here focus on asymmetric anionâÏ catalysis of domino reactions that afford bicyclic products with quaternary stereogenic centers. Catalyst screening includes a newly synthesized, better performing anionâÏ version of classical organocatalysts from cinchona alkaloids, and anionâÏ enzymes. We find stereoselectivities that are clearly better than the best ones reported with conventional catalysts, culminating in unprecedented diastereospecificity. Moreover, we describe achiral salts as supramolecular chirality enhancers and report the first artificial enzyme that operates in neutral water with anionâÏ interactions, i.e., interactions that are essentially new to enzymes. Evidence in support of contributions of anionâÏ interactions to asymmetric catalysis include increasing diastereo- and enantioselectivity with increasing rates, i.e., asymmetric transition-state stabilization in the presence of Ï-acidic surfaces and inhibition with the anion selectivity sequence NO3â > Brâ > BF4â > PF6â
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HaloFlippers: A General Tool for the Fluorescence Imaging of Precisely Localized Membrane Tension Changes in Living Cells.
Tools to image membrane tension in response to mechanical stimuli are badly needed in mechanobiology. We have recently introduced mechanosensitive flipper probes to report quantitatively global membrane tension changes in fluorescence lifetime imaging microscopy (FLIM) images of living cells. However, to address specific questions on physical forces in biology, the probes need to be localized precisely in the membrane of interest (MOI). Herein we present a general strategy to image the tension of the MOI by tagging our newly introduced HaloFlippers to self-labeling HaloTags fused to proteins in this membrane. The critical challenge in the construction of operational HaloFlippers is the tether linking the flipper and the HaloTag: It must be neither too taut nor too loose, be hydrophilic but lipophilic enough to passively diffuse across membranes to reach the HaloTags, and allow partitioning of flippers into the MOI after the reaction. HaloFlippers with the best tether show localized and selective fluorescence after reacting with HaloTags that are close enough to the MOI but remain nonemissive if the MOI cannot be reached. Their fluorescence lifetime in FLIM images varies depending on the nature of the MOI and responds to myriocin-mediated sphingomyelin depletion as well as to osmotic stress. The response to changes in such precisely localized membrane tension follows the validated principles, thus confirming intact mechanosensitivity. Examples covered include HaloTags in the Golgi apparatus, peroxisomes, endolysosomes, and the ER, all thus becoming accessible to the selective fluorescence imaging of membrane tension
Chimeric streptavidins as host proteins for artificial metalloenzymes
The
streptavidin scaffold was expanded with well-structured naturally
occurring motifs. These chimeric scaffolds were tested as hosts for
biotinylated catalysts as artificial metalloenzymes (ArM) for asymmetric
transfer hydrogenation, ring-closing metathesis and anionâÏ
catalysis. The additional second coordination sphere elements significantly
influence both the activity and the selectivity of the resulting hybrid
catalysts. These findings lead to the identification of propitious
chimeric streptavidins for future directed evolution efforts of artificial
metalloenzymes
Optoelectronically mismatched oligophenylethynyl-naphthalenediimide SHJ architectures
The objective of this study was to evaluate the possibility of photoinduced stack/rod electron transfer in surface âzipperâ architectures composed of stacks of blue (B) naphthalenediimides (NDIs) along strings of oligophenylethynyl (OPE) rods. The synthesis and characterization of anionic and cationic multichromophoric OPE-B systems are reported. Absorption spectra suggest that in OPE-B systems, planarity and thus absorption and conductivity of the OPE can possibly be modulated by intramolecular stacking of the surrounding NDIs, although interfering contributions from aggregation remain to be differentiated. Among surface architectures constructed with OPE-B and POP-B systems by zipper and layer-by-layer (LBL) assembly, photocurrents generated by OPE-B zippers exhibit the best critical thickness and fill factors. These findings confirm the existence and functional relevance of topologically matching zipper architectures. In OPE-B zippers, OPEs generate much more photocurrent than the blue NDIs. Ultrafast electron transfer from OPEs to NDIs accounts for these photocurrents, providing wavelength-controlled access to rodâstack charge separation, and thus to formal supramolecular n/p-heterojunctions (SHJs). NDI excitation is not followed by the complementary hole transfer to the OPE rod. Scaffolds with higher HOMOs will be needed to integrate blue NDIs into SHJ photosystems
Correlation of surface pressure and hue of planarizable push-pull chromophores at the air/water interface
It is currently not possible to directly measure the lateral pressure of a biomembrane. Mechanoresponsive fluorescent probes are an elegant solution to this problem but it requires first the establishment of a direct correlation between the membrane surface pressure and the induced color change of the probe. Here, we analyze planarizable dithienothiophene pushâpull probes in a monolayer at the air/water interface using fluorescence microscopy, grazing-incidence angle X-ray diffraction, and infrared reflectionâabsorption spectroscopy. An increase of the lateral membrane pressure leads to a well-packed layer of the âflipperâ mechanophores and a clear change in hue above 18 mN/m. The fluorescent probes had no influence on the measured isotherm of the natural phospholipid DPPC suggesting that the flippers probe the lateral membrane pressure without physically changing it. This makes the flipper probes a truly useful addition to the membrane probe toolbox
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