RESUMEN
Carbon dots (CD) are widely investigated particles with interesting fluorescent properties which are reported to be used for various purposes, as they are biocompatible, resistant to photobleaching and with tuneable properties depending on the specific CD surface chemistry. In this work, we report on the possibility to use opportunely designed CD to distinguish among isobaric peptides almost undistinguishable by mass spectrometry, as well as to monitor protein aggregation phenomena. Particularly, cell-penetrating peptides containing the carnosine moiety at different positions in the peptide chain produce sequence specific fluorescent signals. Analogously, different insulin oligomerization states can also be distinguished by the newly proposed experimental approach. The latter is here described in details and can be potentially applied to any kind of peptide or protein.
RESUMEN
Every biological and physicochemical process occurring in a fluid phase depends on the diffusion coefficient (D) of the species in solution. In the present work, a model to describe and fit the behaviour of D as a function of structure and extensive thermodynamics parameters in binary solutions of linear chain organic molecules is developed. Supporting experimental and computational evidences for this model are obtained by measuring D for a series of n-alcohols through a novel surface plasmon resonance method and molecular dynamics simulations. This allows to propose a kind of combined analysis to explain the dependence of D on various thermodynamic and structural parameters. The results suggest that for small linear systems in the range from 0 to 200 g mol-1 and under the assumption that the diffusive activation energy is a linear function of mass, D is strictly dependent on the molecular shape and on the relative strength of the solute-solvent intermolecular forces represented by a parameter named R. The newly proposed approach can be utilized to characterize and monitor progressive changes in physicochemical properties for any investigated species upon increasing the dimension of the aggregate/molecule along a certain direction.
RESUMEN
Poorly water-soluble photosensitive monolayers might enable very precise control of the rate and number of desorbing molecules by controlling both the monolayer density and conformational composition. In this perspective, we systematically characterized the interfacial behavior of Langmuir monolayers consisting of a poorly water-soluble azobenzene-containing surfactant as a function of its trans/cis ratio. Precise control of the conformational ratio was achieved by controlling the UV irradiation time, allowing researchers to investigate compositions spanning from 100% trans to 90% cis. Our results demonstrate that in 100% trans monolayers, molecules do not desorb with compression until a threshold area is reached. Instead, the number of molecules desorbing in mixed trans-cis monolayers can be modulated by controlling both the composition and the compression rate. Additionally, the desorption rate at constant density is also strongly composition-dependent, and it accounts for two different regimes with two different characteristic times. We will show that trans molecules mostly desorb according to the slow regime while cis molecules conform to the fast one, but the two conformers mutually influence each other.
RESUMEN
The possibility to monitor peptide and protein aggregation is of paramount importance in the so-called conformational diseases, as the understanding of many physiological pathways, as well as pathological processes involved in the development of such diseases, depends very much on the actual possibility to monitor biomolecule oligomeric distribution and aggregation. In this work, we report a novel experimental method to monitor protein aggregation, based on the change of the fluorescent properties of carbon dots upon protein binding. The results obtained in the case of insulin with this newly proposed experimental approach are compared with those obtained with other common experimental techniques normally used for the same purpose (circular dichroism, DLS, PICUP and ThT fluorescence). The greatest advantage of the hereby presented methodology over all the other experimental methods considered is the possibility to monitor the initial stages of insulin aggregation under the different experimental conditions sampled and the absence of possible disturbances and/or molecular probes during the aggregation process.
Asunto(s)
Insulina , Puntos Cuánticos , Insulina/química , Carbono/química , Agregado de Proteínas , Puntos Cuánticos/química , Dicroismo Circular , Colorantes Fluorescentes/químicaRESUMEN
Insulin is commonly used to treat diabetes and undergoes aggregation at the site of repeated injections in diabetic patients. Moreover, aggregation is also observed during its industrial production and transport and should be avoided to preserve its bioavailability to correctly adjust glucose levels in diabetic patients. However, monitoring the effect of various parameters (pH, protein concentration, metal ions, etc.) on the insulin aggregation and oligomerization state is very challenging. In this work, we have applied a novel Surface Plasmon Resonance (SPR)-based experimental approach to insulin solutions at various experimental conditions, monitoring how its diffusion coefficient is affected by pH and the presence of metal ions (copper and zinc) with unprecedented sensitivity, precision, and reproducibility. The reported SPR method, hereby applied to a protein for the first time, besides giving insight into the insulin oligomerization and aggregation phenomena, proved to be very robust for determining the diffusion coefficient of any biomolecule. A theoretical background is given together with the software description, specially designed to fit the experimental data. This new way of applying SPR represents an innovation in the bio-sensing field and expanding the potentiality of commonly used SPR instruments well over the canonical investigation of biomolecular interactions.
Asunto(s)
Técnicas Biosensibles , Diabetes Mellitus , Humanos , Resonancia por Plasmón de Superficie/métodos , Insulina/química , Reproducibilidad de los Resultados , Metales , Iones , Técnicas Biosensibles/métodosRESUMEN
Glaucoma represents a group of progressive neurodegenerative diseases characterized by the loss of retinal ganglion cells (RGCs) and their axons with subsequent visual field impairment. The disease develops through largely uncharacterized molecular mechanisms, that are likely to occur in different localized cell types, either in the anterior (e.g., trabecular meshwork cells) or posterior (e.g., Muller glia, retinal ganglion cells) segments of the eye. Genomic and preclinical studies suggest that glaucoma pathogenesis may develop through altered ubiquitin (Ub) signaling. Ubiquitin conjugation, referred to as ubiquitylation, is a major post-synthetic modification catalyzed by E1-E2-E3 enzymes, that profoundly regulates the turnover, trafficking and biological activity of the targeted protein. The development of new technologies, including proteomics workflows, allows the biology of ubiquitin signaling to be described in health and disease. This post-translational modification is emerging as a key role player in neurodegeneration, gaining relevance for novel therapeutic options, such as in the case of Proteolysis Targeting Chimeras technology. Although scientific evidence supports a link between Ub and glaucoma, their relationship is still not well-understood. Therefore, this review provides a detailed research-oriented discussion on current evidence of Ub signaling in glaucoma. A review of genomic and genetic data is provided followed by an in-depth discussion of experimental data on ASB10, parkin and optineurin, which are proteins that play a key role in Ub signaling and have been associated with glaucoma.