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 + . Here, we show that K + channels remain permeable to water, even after entering such an electrically silent conformation. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K + channel KcsA into lipid vesicles that were either C-type inactivating or noninactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. Quantification of the per-channel increment in proteoliposome water permeability with the aid of stopped-flow experiments yielded a unitary water permeability p f of (6.9 ± 0.6) × 10 −13 cm 3 ·s −1 for both mutants. "Collapse" of the selectivity filter upon K + removal did not alter p f and was fully reversible, as demonstrated by current measurements through planar bilayers in a K + -containing medium to which K + -free proteoliposomes were fused. Water flow through KcsA is halved by 200 mM K + in the aqueous solution, which indicates an effective K + dissociation constant in that range for a singly occupied channel. This questions the widely accepted hypothesis that multiple K + ions in the selectivity filter act to mutually destabilize binding. membrane channels | protein reconstitution | knock-on mechanism | aquaporin | brain water homeostasis

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). Thermodynamic parameters of vesicle solubilization and reconstitution, membrane partitioning, and micelle formation were assessed by right-angle light scattering and isothermal titration calorimetry. Membrane translocation and dissolution proceed very slowly at 25 degrees C but are considerably accelerated at 65 degrees C. At this temperature, a simple SDS/POPC phase diagram (comprising vesicular, coexistence, and micellar ranges) and a complete set of partition coefficients and transfer enthalpies were obtained. Electrostatic repulsion effects at the membrane surface were implemented by combining Gouy-Chapman theory with a Langmuir adsorption isotherm to account for Na+ binding to membrane-incorporated DS-. This approach offered a quantitative understanding of solubilization and reconstitution processes, which were interpreted in terms of partition equilibria between and ideal mixing in all phases. More than any other property, the transbilayer flip-flop rate under given experimental conditions hence appears to dictate a detergent's suitability for thermodynamically controlled lipid membrane solubilization and reconstitution.

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. Even at starting concentrations in the low micromolar range, KcsA has a tremendous impact on the supramolecular organization of the system, shifting the critical lipid/detergent ratios at the onset and completion of vesicle formation by more than 2-fold. Thus, equilibrium phase diagrams obtained for protein-free lipid/detergent mixtures would be misleading when used to guide the reconstitution process. To address this issue, we demonstrate that, even under such nonequilibrium conditions, high-sensitivity isothermal titration calorimetry can be exploited to monitor the progress of membrane-protein reconstitution in real time, in a noninvasive manner, and at high resolution to yield functional proteoliposomes with a narrow size distribution for further downstream applications.

α-Synuclein (α-Syn) liquid-liquid phase separation (LLPS) leads to irreversible amyloid fibril formation associated with Parkinson's disease pathogenesis. Critical concentrations of α-Syn LLPS are relatively high under physiological solution conditions. Moreover, α-Syn exhibits delayed LLPS kinetics under certain conditions which deviates from the behaviour predicted by classical homogeneous nucleation theory. In the current body of work, using interferometric light scattering (iSCAT), also known as mass photometry, we experimentally probe that α-Syn can form nanoscale phase separated assemblies/clusters, containing tens to hundreds of moleculesboth above and below the critical LLPS concentration down to physiologically relevant scales. The formation of these clusters is instantaneous, even under conditions where the formation of microscopically visible droplets takes several days. However, they account for a very small volume fraction below saturation concentration. The slow growth of the nanoclusters can be attributed to a kinetic barrier which can be overcome by increasing the solution temperature to just below the droplet melting point. We provide reasons for caution in quantifying dilute phase concentrations for α-Syn LLPS samples containing nanoscale droplets-which can only be separated using ultracentrifugation. In addition, we also delineate that the presence of certain surfaces facilitates α-Syn droplet nucleation under conditions of delayed kinetics but is not a mandatory prerequisite for nanocluster formation. Taken together, our findings reveal that phase separation of α-Syn occurs at a wider range of solution conditions than predicted so far and provides an important step towards understanding α-Syn LLPS within physiological scales..

α-Synuclein (α-Syn) liquid-liquid phase separation (LLPS) leads to irreversible amyloid fibril formation associated with Parkinsons disease pathogenesis. Critical concentrations of α-Syn LLPS are relatively high under physiological solution conditions. Moreover, α-Syn exhibits delayed LLPS kinetics under certain conditions which deviates from the behaviour predicted by classical homogeneous nucleation theory. In the current body of work, using interferometric light scattering (iSCAT), also known as mass photometry, we experimentally probe that α-Syn can form nanoscale phase separated assemblies/clusters, containing tens to hundreds of molecules, both above and below the critical LLPS concentration down to physiologically relevant scales. The formation of these clusters is instantaneous, even under conditions where the formation of microscopically visible droplets takes several days. However, they account for a very small volume fraction below saturation concentration. The slow growth of the nanoclusters can be attributed to a kinetic barrier which can be overcome by increasing the solution temperature to just below the droplet melting point. We provide reasons for caution in quantifying dilute phase concentrations for α-Syn LLPS samples containing nanoscale droplets, which can only be separated using ultracentrifugation. In addition, we also delineate that the presence of certain surfaces facilitates α-Syn droplet nucleation under conditions of delayed kinetics but is not a mandatory prerequisite for nanocluster formation. Taken together, our findings reveal that phase separation of α-Syn occurs at a wider range of solution conditions than predicted so far and provides an important step towards understanding α-Syn LLPS within physiological scales.

No abstract

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. A mass spectrometric proteomics approach that combines domain-based pulldown experiments, binding site inactivation, and stable isotope labeling by amino acids in cell culture (SILAC) was then used to delineate the relative importance of the peptide and ubiquitin binding sites.

Membrane protein reconstitution denotes the transfer of a purified (but usually inactive) membrane protein from detergent micelles into lipid bilayers. The aim is to restore the native protein fold and function in a well-defined membrane environment. The reconstitution yield critically depends on a wide range of parameters, including temperature, pH, ionic strength, as well as the type and concentration of detergent, lipid, protein, and additives. Moreover, it is of paramount importance to initiate the reconstitution process from a suitable lipid-to-detergent ratio. Unfortunately, however, assessing the success of a reconstitution experiment has thus far been limited to a trial-and-error approach, which has substantially slowed progress in the field.To address this problem, we have established high-sensitivity isothermal titration calorimetry (ITC) as a powerful tool for monitoring the reconstitution of membrane proteins into lipid vesicles. Using ITC, the complex changes in the physical state of a protein/lipid/detergent mixture during reconstitution can be followed in a non-invasive and fully automated manner. Here we exemplify this approach for the prokaryotic potassium channel KcsA, which we first purified in detergent micelles and then reconstituted into stable proteoliposomes at very high protein densities. Electrophysiological experiments performed in planar lipid membranes confirmed that KcsA regained its functional activity upon reconstitution.