RESUMEN
Polymer microgels are swollen macromolecular networks with a typical size of hundred of nanometers to several microns that show an extraordinary open and responsive architecture to different external stimuli, being therefore important candidates for nanobiotechnology and nanomedical applications such as biocatalysis, sensing and drug delivery. It is therefore crucial to understand the delicate balance of physical-chemical interactions between the polymer backbone and solvent molecules that to a high extent determine their responsivity. In particular, the co-nonsolvency effect of poly(N-isopropylacrylamide) in aqueous alcohols is highly discussed, and there is a disagreement between molecular dynamics (MD) simulations (from literature) of the preferential adsorption of alcohol on the polymer chains and the values obtained by several empirical methods that mostly probe the bulk solvent properties. It is our contention that the most efficacious method for addressing this problem requires a nanoscopic method that can be combined with spectroscopy and record fluorescence spectra and super-resolved fluorescence lifetime images of microgels labeled covalently with the solvatochromic dye Nile Red. By employing this approach, we could simultaneously resolve the structure of sub-micron size objects in the swollen and in the collapsed state and estimate the solvent composition inside of them in - mixtures for two very different polymer architectures. We found an outstanding agreement between the MD simulations and our results that estimate a co-solvent molar fraction excess of approximately 3 with a very flat profile in the lateral direction of the microgel.
RESUMEN
Herein, we show that a far-red arylidene-squaraine dye is stable against nucleophiles, in contrast to arene-squaraines. Owing to the fluorescence enhancement in apolar media together with high brightness and photostability, this dye was successfully applied to detect the oxytocin G protein-coupled receptor and monitor its internalization in living cells.
Asunto(s)
Ciclobutanos/química , Colorantes Fluorescentes/química , Imagen Molecular , Fenoles/química , Receptores de Oxitocina/química , Receptores de Oxitocina/metabolismo , Animales , Bovinos , Color , Células HEK293 , Humanos , Transporte de ProteínasRESUMEN
Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide-membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.
Asunto(s)
Péptidos/química , Polilisina/química , Péptidos Catiónicos Antimicrobianos/química , Biofisica/métodos , Relación Dosis-Respuesta a Droga , Flavonoides/química , Colorantes Fluorescentes/farmacología , Lípidos/química , Magaininas , Espectroscopía de Resonancia Magnética/métodos , Meliteno/química , Modelos Estadísticos , Unión Proteica , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia/métodos , Espectrofotometría/métodos , Proteínas de Xenopus/químicaRESUMEN
Bovine eye lens alpha-crystallin was covalently labeled with 6-bromomethyl-4'-diethylamino-3-hydroxyflavone and studied under native-like conditions and at the elevated temperature (60 degrees C) that is known to facilitate alpha-crystallin chaperone-like activity. This novel SH-reactive two-band ratiometric fluorescent probe is characterized by two highly emissive N*- and T*-bands; the latter appears due to excited state intramolecular proton transfer reaction. The positions of these bands and the ratio of their intensities for the alpha-crystallin-dye conjugate are the sensitive indicators of polarity of the dye environment and its participation in intermolecular hydrogen bonding. Although we found that the dye labels both the SH and the NH2 groups in alpha-crystallin, a recently developed procedure allowed us to distinguish between the heat-induced spectral changes of the dye molecules attached to SH and NH2 groups. We observed that at elevated temperature the environment of the SH-attached dye becomes more polar and flexible. The number of H-bond acceptor groups in the vicinity of the dye decreases. Since alpha-crystallin contains a single Cys residue within the C-terminal domain of its (alpha)A subunit (the (alpha)B subunit contains none), we can attribute the observed effects to temperature-induced changes in the C-terminal domain of this protein.