Enhancing RNA encapsulation quantification in lipid nanoparticles: Sustainable alternatives to Triton X-100 in the RiboGreen assay.

To quantify concentration and encapsulation efficiency (EE) of mRNA in lipid nanoparticles (LNPs) the RiboGreen assay is extensively used. As part of this assay, a surfactant is used to release mRNA from LNPs for detection with the RiboGreen dye. So far, the surfactant of choice has been Triton X-100, which is harmful to human health and the environment.

Mass photometric detection and quantification of nanoscale α-synuclein phase separation.

Protein liquid-liquid phase separation can lead to disease-related amyloid fibril formation. The mechanisms of conversion of monomeric protein into condensate droplets and of the latter into fibrils remain elusive. Here, using mass photometry, we demonstrate that the Parkinson's disease-related protein, α-synuclein, can form dynamic nanoscale clusters at physiologically relevant, sub-saturated concentrations.

Real-time monitoring of membrane-protein reconstitution by isothermal titration calorimetry.

Phase diagrams offer a wealth of thermodynamic information on aqueous mixtures of bilayer-forming lipids and micelle-forming detergents, providing a straightforward means of monitoring and adjusting the supramolecular state of such systems. However, equilibrium phase diagrams are of very limited use for the reconstitution of membrane proteins because of the occurrence of irreversible, unproductive processes such as aggregation and precipitation that compete with productive reconstitution. Here, we exemplify this by dissecting the effects of the K(+) channel KcsA on the process of bilayer self-assembly in a mixture of Escherichia coli polar lipid extract and the nonionic detergent octyl-β-d-glucopyranoside.

Filter gate closure inhibits ion but not water transport through potassium channels.

The selectivity filter of K(+) channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K(+) hydration. Ion conductivity is abrogated if some of these carbonyl groups flip out of the lumen, which happens (i) in the process of C-type inactivation or (ii) during filter collapse in the absence of K(+).

Membrane solubilisation and reconstitution by octylglucoside: comparison of synthetic lipid and natural lipid extract by isothermal titration calorimetry.

We have studied the solubilisation and reconstitution of lipid membranes composed of either synthetic phosphatidylcholine or Escherichia. coli polar lipid extract by the non-ionic detergent octylglucoside. For both lipid systems, composition-dependent transformations of unilamellar vesicles into micelles or vice versa were followed by high-sensitivity isothermal titration calorimetry.

Proline-rich sequence recognition: II. Proteomics analysis of Tsg101 ubiquitin-E2-like variant (UEV) interactions.

The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. The ubiquitin-E2-like variant (UEV) domain of the protein interacts with proline-rich sequences of target proteins that contain P(S/T)AP amino acid motifs and weakly binds to the ubiquitin moiety of proteins committed to sorting or degradation. Here we performed peptide spot analysis and phage display to refine the peptide binding specificity of the Tsg101 UEV domain.

Thermodynamics of lipid membrane solubilization by sodium dodecyl sulfate.

We provide a comprehensive thermodynamic description of lipid membrane dissolution by a charged detergent. To this end, we have studied the interactions between the anionic detergent sodium dodecyl sulfate (SDS) and the zwitterionic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in dilute aqueous solution (10 mM phosphate buffer, 154 mM NaCl, pH 7.4).