RESUMEN
The serotonergic transmitter system plays fundamental roles in the nervous system in neurotransmission, synaptic plasticity, pathological processes, and therapeutic effects of antidepressants and psychedelics, as well as in the gastrointestinal and circulatory systems. We introduce a novel small molecule fluorescent agent, termed SERTlight, that specifically labels serotonergic neuronal cell bodies, dendrites, and axonal projections as a serotonin transporter (SERT) fluorescent substrate. SERTlight was developed by an iterative molecular design process, based on an aminoethyl-quinolone system, to integrate structural elements that impart SERT substrate activity, sufficient fluorescent brightness, and a broad absence of pharmacological activity, including at serotonin (5-hydroxytryptamine, 5HT) receptors, other G protein-coupled receptors (GPCRs), ion channels, and monoamine transporters. The high labeling selectivity is not achieved by high affinity binding to SERT itself but rather by a sufficient rate of SERT-mediated transport of SERTlight, resulting in accumulation of these molecules in 5HT neurons and yielding a robust and selective optical signal in the mammalian brain. SERTlight provides a stable signal, as it is not released via exocytosis nor by reverse SERT transport induced by 5HT releasers such as MDMA. SERTlight is optically, pharmacologically, and operationally orthogonal to a wide range of genetically encoded sensors, enabling multiplexed imaging. SERTlight enables labeling of distal 5HT axonal projections and simultaneous imaging of the release of endogenous 5HT using the GRAB5HT sensor, providing a new versatile molecular tool for the study of the serotonergic system.
Asunto(s)
Colorantes Fluorescentes , Serotonina , Animales , Serotonina/metabolismo , Colorantes Fluorescentes/metabolismo , Neuronas/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Encéfalo/metabolismo , Mamíferos/metabolismoRESUMEN
Carbohydrates are common co-solutes for the stabilization of proteins. The effect of carbohydrate solutions on the stability of collagen, the most abundant protein in mammals, is, however, underexplored. In this work, we studied the thermal stability of collagen triple helices derived from a molecularly defined collagen model peptide (CMP), Ac-(Pro-Hyp-Gly)7 -NH2 , in solutions of six common mono- and disaccharides. We show that the carbohydrates stabilize the collagen triple helix in a concentration-dependent manner, with an increase of the melting temperature of up to 17 °C. In addition, we show that the stabilizing effect is similar for all studied sugars, including trehalose, which is otherwise considered a privileged bioprotectant. The results provided insight into the effects of sugar co-solutes on collagen triple helices and can aid the selection of storage environments for collagen-based materials and probes.
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Colágeno , Disacáridos , Animales , Temperatura , Trehalosa , MamíferosRESUMEN
Nature uses elaborate methods to control protein assembly, including that of heterotrimeric collagen. Here, we established design principles for the composition and register-selective assembly of synthetic collagen heterotrimers. The assembly code enabled the self-sorting of eight different strands into threeâout of 512 possibleâtriple helices via complementary (4S)-aminoproline and aspartate residues. Native ESI-MS corroborated the specific assembly into coexisting heterotrimers.
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Ácido Aspártico , Colágeno , Multimerización de Proteína , Colágeno/química , Movimiento CelularRESUMEN
N-terminal acylation is a common tool for the installation of functional moieties (e.g., sensors or bioactive molecules) on collagen model peptides (CMPs). The N-acyl group and its length are generally assumed to have little or no influence on the properties of the collagen triple helix formed by the CMP. Here, we show that the length of short (C1-C4) acyl capping groups has different effects on the thermal stability of collagen triple helices in POG, OGP, and GPO frames. While the effect of different capping groups on the stability of triple helices in the GPO frame is negligible, longer acyl chains stabilize OGP triple helices but destabilize POG analogues. The observed trends arise from a combination of steric repulsion, the hydrophobic effect, and n â π* interactions. Our study provides a basis for the design of N-terminally functionalized CMPs with predictable effects on triple helix stability.
Asunto(s)
Colágeno , Péptidos , Colágeno/química , Péptidos/química , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Collagen model peptides (CMPs) consisting of proline-(2S,4R)-hydroxyproline-glycine (POG) repeats have provided a breadth of knowledge of the triple helical structure of collagen, the most abundant protein in mammals. Predictive tools for triple helix stability have, however, lagged behind since the effect of CMPs with different frames ([POG]n , [OGP]n , or [GPO]n ) and capped or uncapped termini have so far been underestimated. Here, we elucidated the impact of the frame, terminal functional group and its charge on the stability of collagen triple helices. Combined experimental and theoretical studies with frame-shifted, capped and uncapped CMPs revealed that electrostatic interactions, strand preorganization, interstrand H-bonding, and steric repulsion at the termini contribute to triple helix stability. We show that these individual contributions are additive and allow for the prediction of the melting temperatures of CMP trimers.
Asunto(s)
Colágeno , Péptidos , Animales , Colágeno/química , Péptidos/química , Prolina/química , Hidroxiprolina/química , Glicina , MamíferosRESUMEN
Collagen model peptides (CMPs), composed of proline-(2S,4R)-hydroxyproline-glycine (POG) repeat units, have been extensively used to study the structure and stability of triple-helical collagenâthe dominant structural protein in mammalsâat the molecular level. Despite the more than 50-year history of CMPs and numerous studies on the relationship between the composition of single-stranded CMPs and the thermal stability of the assembled triple helices, little attention has been paid to the effects arising from their terminal residues. Here, we show that frame-shifted CMPs, which share POG repeat units but terminate with P, O, or G, form triple helices with vastly different thermal stabilities. A melting temperature difference as high as 16 °C was found for triple helices from 20-mers Ac-OG[POG]6-NH2 and Ac-[POG]6PO-NH2, and triple helices of the constitutional isomers Ac-[POG]7-NH2 and Ac-[GPO]7-NH2 melt 10 °C apart. A combination of thermal denaturation, circular dichroism and NMR spectroscopic studies, and molecular dynamics simulations revealed that the stability differences originate from the propensity of the peptide termini to preorganize into a polyproline-II helical structure. Our results advise that care must be taken when designing peptide mimics of structural proteins, as subtle changes in the terminal residues can significantly affect their properties. Our findings also provide a general and straightforward tool for tuning the stability of CMPs for applications as synthetic materials and biological probes.
Asunto(s)
Colágeno , Péptidos , Secuencia de Aminoácidos , Dicroismo Circular , Colágeno/química , Glicina , Hidroxiprolina/química , Péptidos/química , Prolina/químicaRESUMEN
Optical imaging of changes in the membrane potential of living cells can be achieved by means of fluorescent voltage-sensitive dyes (VSDs). A particularly challenging task is to efficiently deliver these highly lipophilic probes to specific neuronal subpopulations in brain tissue. We have tackled this task by designing a solubilizing, hydrophilic polymer platform that carries a high-affinity ligand for a membrane protein marker of interest and a fluorescent VSD. Here, we disclose an improved design of polymer-supported probes for chemical, nongenetic targeting of voltage sensors to axons natively expressing the dopamine transporter in ex vivo mouse brain tissue. We first show that for negatively charged rhodol VSDs functioning on the photoinduced electron transfer principle, poly(ethylene glycol) as a carrier enables targeting with higher selectivity than the polysaccharide dextran in HEK cell culture. In the same experimental setting, we also demonstrate that incorporation of an azetidine ring into the rhodol chromophore substantially increases the brightness and voltage sensitivity of the respective VSD. We show that the superior properties of the optimized sensor are transferable to recording of electrically evoked activity from dopaminergic axons in mouse striatal slices after averaging of multiple trials. Finally, we suggest the next milestones for the field to achieve single-scan recordings with nongenetically targeted VSDs in native brain tissue.
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Neuronas Dopaminérgicas , Colorantes Fluorescentes , Animales , Colorantes Fluorescentes/química , Potenciales de la Membrana/fisiología , Ratones , Polímeros , XantonasRESUMEN
Mitragynine (MG) is the most abundant alkaloid component of the psychoactive plant material "kratom", which according to numerous anecdotal reports shows efficacy in self-medication for pain syndromes, depression, anxiety, and substance use disorders. We have developed a synthetic method for selective functionalization of the unexplored C11 position of the MG scaffold (C6 position in indole numbering) via the use of an indole-ethylene glycol adduct and subsequent iridium-catalyzed borylation. Through this work we discover that C11 represents a key locant for fine-tuning opioid receptor signaling efficacy. 7-Hydroxymitragynine (7OH), the parent compound with low efficacy on par with buprenorphine, is transformed to an even lower efficacy agonist by introducing a fluorine substituent in this position (11-F-7OH), as demonstrated in vitro at both mouse and human mu opioid receptors (mMOR/hMOR) and in vivo in mouse analgesia tests. Low efficacy opioid agonists are of high interest as candidates for generating safer opioid medications with mitigated adverse effects.
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Mitragyna/química , Extractos Vegetales/farmacología , Receptores Opioides mu/agonistas , Alcaloides de Triptamina Secologanina/farmacología , Analgésicos/química , Analgésicos/farmacología , Animales , Glicol de Etileno/química , Humanos , Ratones Noqueados , Modelos Químicos , Estructura Molecular , Extractos Vegetales/química , Unión Proteica , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Alcaloides de Triptamina Secologanina/químicaRESUMEN
The bark beetle Phloeotribus rhododactylus feeds mainly on the shrub Cytisus scoparius. The range of P. rhododactylus extends from Spain in the south to southern Sweden, Denmark, and Scotland in the north. Its range to the east extends to Poland, Slovakia, and Hungary, but single localities are known further east in Romania, Bulgaria, and Greece. It is clear that the range of the beetle matches that of its main host. C. scoparius is adapted to Mediterranean and coastal climates, and its range is limited by low winter temperatures. P. rhododactylus is, therefore, rare in Central Europe. It infests either individuals of C. scoparius that have been damaged by mammalian herbivores or snow or that are drought-stressed. Although C. scoparius is an invasive plant in agricultural and natural ecosystems, P. rhododactylus has not been found in any of the areas where C. scoparius has invaded.
RESUMEN
Voltage sensitive fluorescent dyes (VSDs) are important tools for probing signal transduction in neurons and other excitable cells. The impact of these highly lipophilic sensors has, however, been limited due to the lack of cell-specific targeting methods in brain tissue or living animals. We address this key challenge by introducing a nongenetic molecular platform for cell- and molecule-specific targeting of synthetic VSDs in the brain. We employ a dextran polymer particle to overcome the inherent lipophilicity of VSDs by dynamic encapsulation and high-affinity ligands to target the construct to specific neuronal cells utilizing only native components of the neurotransmission machinery at physiological expression levels. Dichloropane, a monoamine transporter ligand, enables targeting of dense dopaminergic axons in the mouse striatum and sparse noradrenergic axons in the mouse cortex in acute brain slices. PFQX in conjunction with ligand-directed acyl imidazole chemistry enables covalent labeling of AMPA-type glutamate receptors in the same brain regions. Probe variants bearing either a classical electrochromic ANEP dye or state-of-the-art VoltageFluor-type dye respond to membrane potential changes in a similar manner to the parent dyes, as shown by whole-cell patch recording. We demonstrate the feasibility of optical voltage recording with our probes in brain tissue with one-photon and two-photon fluorescence microscopy and define the signal limits of optical voltage imaging with synthetic sensors under a low photon budget determined by the native expression levels of the target proteins. This work demonstrates the feasibility of a chemical targeting approach and expands the possibilities of cell-specific imaging and pharmacology.
Asunto(s)
Encéfalo , Cocaína/análogos & derivados , Dopamina/análisis , Colorantes Fluorescentes/química , Norepinefrina/análisis , Animales , Encéfalo/citología , Cocaína/síntesis química , Cocaína/química , Colorantes Fluorescentes/síntesis química , Ratones , Microscopía Fluorescente , Modelos Moleculares , Estructura Molecular , Imagen ÓpticaRESUMEN
Driving forces of anion binding in water in contrast to nonpolar environments are of high interest because of their relevance to biology and medicine. Here we report a neutral bambusuril macrocycle (1), soluble in both water and nonpolar solvents due to decoration with 12 polyethylene glycol-based substituents. The new bambusuril has the highest affinity for I- in pure water ever reported for a synthetic macrocycle relying on hydrogen bonding interactions rather than metal coordination or Coulombic forces. Isothermal titration calorimetry (ITC) experiments in nine different solvents, ranging from polar water to nonpolar carbon tetrachloride, provided insight into the forces responsible for halide binding by bambusurils. The different importance of anion solvation and solvent expulsion from the cavity of the macrocycle in various solvents is illustrated by the fact that halide binding in water and chloroform is exclusively driven by favorable enthalpy with an entropic penalty, while in alcohols and nonpolar solvents, both favorable enthalpy and entropy contribute to anion encapsulation. DFT calculations and correlation of thermodynamic data with the solvent Swain acity parameter further underscore the importance of solvent effects on anion binding by bambusurils.
RESUMEN
Methyl viologen hexafluorophosphate (MV2+·2PF6-) and dodecamethylbambus[6]uril (BU6) form crystals in which the layers of viologen dications alternate with those of a 1:2 supramolecular complex of BU6 and PF6-. This arrangement allows for a one-electron reduction of MV2+ ions upon UV irradiation to form MV+⢠radical cations within the crystal structure with half-lives of several hours in air. The mechanism of this photoinduced electron transfer in the solid state and the origin of the long-lived charge-separated state were studied by steady-state and transient spectroscopies, cyclic voltammetry, and electron paramagnetic resonance spectroscopy. Our experiments are supported by quantum-chemical calculations showing that BU6 acts as a reductant. In addition, analogous photochemical behavior is also demonstrated on other MV2+/BU6 crystals containing either BF4- or Br- counterions.