Channel geometry governs the unitary osmotic water channel permeability, p_f, according to classical hydrodynamics. Yet, p_f varies by several orders of magnitude for membrane channels with a constriction zone that is one water molecule in width and four to eight molecules in length. We show that both the p_f of those channels and the diffusion coefficient of the single-file waters within them are determined by the number N_H of residues in the channel wall that may form a hydrogen bond with the single-file waters. The logarithmic dependence of water diffusivity on N_H is in line with the multiplicity of binding options at higher N_H densities. We obtained high-precision p_f values by (i) having measured the abundance of the reconstituted aquaporins in the vesicular membrane via fluorescence correlation spectroscopy and via high-speed atomic force microscopy, and (ii) having acquired the vesicular water efflux from scattered light intensities via our new adaptation of the Rayleigh-Gans-Debye equation.

After a short introduction into the single-file transport theory, we analyze experiments in which the unitary water permeability, p_f, of water channel proteins (aquaporins, AQPs), potassium channels (KcsA), and antibiotics (gramicidin-A derivatives) has been obtained. A short outline of the underlying methods is also provided.

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.

Background: Lipid-protein interactions stabilize protein oligomers, shape their structure, and modulate their function. Whereas in vitro experiments already account for the functional importance of lipids by using natural lipid extracts, in silico methods lack behind by embedding proteins in single component lipid bilayers. However, to accurately complement in vitro experiments with molecular details at very high spatio-temporal resolution, molecular dynamics simulations have to be performed in natural(-like) lipid environments. Results: To enable more accurate MD simulations, we have prepared four membrane models of E. coli polar lipid extract, a typical model organism, each at all-atom (CHARMM36) and coarse-grained (Martini3) representations. These models contain all main lipid headgroup types of the E. coli inner membrane, i.e., phosphatidylethanolamines, phosphatidylglycerols, and cardiolipins, symmetrically distributed between the membrane leaflets. The lipid tail (un)saturation and propanylation stereochemistry represent the bacterial lipid tail composition of E. coli grown at 37^∘C until 3/4 of the log growth phase. The comparison of the Simple three lipid component models to the complex 14-lipid component model Avanti over a broad range of physiologically relevant temperatures revealed that the balance of lipid tail unsaturation and propanylation in different positions and inclusion of lipid tails of various length maintain realistic values for lipid mobility, membrane area compressibility, lipid ordering, lipid volume and area, and the bilayer thickness. The only Simple model that was able to satisfactory reproduce most of the structural properties of the complex Avanti model showed worse agreement of the activation energy of basal water permeation with the here performed measurements. The Martini3 models reflect extremely well both experimental and atomistic behavior of the E. coli polar lipid extract membranes. Aquaporin-1 embedded in our native(-like) membranes causes partial lipid ordering and membrane thinning in its vicinity. Moreover, aquaporin-1 attracts and temporarily binds negatively charged lipids, mainly cardiolipins, with a distinct cardiolipin binding site in the crevice at the contact site between two monomers, most probably stabilizing the tetrameric protein assembly. Conclusions: The here prepared and validated membrane models of E. coli polar lipids extract revealed that lipid tail complexity, in terms of double bond and cyclopropane location and varying lipid tail length, is key to stabilize membrane properties over a broad temperature range. In addition, they build a solid basis for manifold future simulation studies on more realistic lipid membranes bridging the gap between simulations and experiments.

Membrane interleaflet viscosity ${\eta }_e$ affects tether formation, phase separation into domains, cell shape changes, and budding. Contrary to the expected contribution to interleaflet coupling from interdigitation, the slide of lipid patches in opposing monolayers conferred the same value ${\eta }_e\approx 3\times {10}^9\mathrm{J}\mathrm{s}{\mathrm{m}}^{-4}$ for the friction experienced by the ends of both short and long chain fluorescent lipid analogues. Consistent with the weak dependence of the translational diffusion coefficient on lipid length, the in-layer viscosity was, albeit length dependent, much smaller than ${\eta }_e$.