Visualization of the mechanosensitive ion channel MscS under membrane tension.

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes1-3. However, how exactly they sense mechanical force remains under investigation4. The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels4-8, but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states9-11. Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tension. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformation of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensation. Pore lipids are necessary to prevent ion conduction in the closed state. Gatekeeper lipids stabilize the closed conformation and dissociate with membrane tension, allowing the channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tension, allowing the channel to transition to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensation4,11.

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays.

Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillar[5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(± 0.3) × 10(-14) cm(3)/s or 3.5(± 1.0) × 10(8) water molecules per s, which is in the range of AQPs (3.4 ∼ 40.3 × 10(8) water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 10(8) water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼ 10(7) water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼ 2.6 × 10(5) pores per µm(2)) is two orders of magnitude higher than that of CNT membranes (0.1 ∼ 2.5 × 10(3) pores per µm(2)). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays.

Magnetically Directed Two-Dimensional Crystallization of OmpF Membrane Proteins in Block Copolymers.

Two-dimensional (2D) alignment and crystallization of membrane proteins (MPs) is increasingly important in characterizing their three-dimensional (3D) structure, in designing pharmacological agents, and in leveraging MPs for biomimetic devices. Large, highly ordered MP 2D crystals in block copolymer (BCP) matrices are challenging to fabricate, but a facile and scalable technique for aligning and crystallizing MPs in thin-film geometries would rapidly translate into applications. This work introduces a novel method to grow larger and potentially better ordered 2D crystals by performing the crystallization process in the presence of a strong magnetic field. We demonstrate the efficacy of this approach using a ß-barrel MP, outer membrane protein F (OmpF), in short-chain polybutadiene-poly(ethylene oxide) (PB-PEO) membranes. Crystals grown in a magnetic field were up to 5 times larger than conventionally grown crystals, and a signal-to-noise (SNR) analysis of diffraction peaks in Fourier transforms of specimens imaged by negative-stain electron microscopy (EM) and cryo-EM showed twice as many high-SNR diffraction peaks, indicating that the magnetic field also improves crystal order.

High-density reconstitution of functional water channels into vesicular and planar block copolymer membranes.

The exquisite selectivity and unique transport properties of membrane proteins can be harnessed for a variety of engineering and biomedical applications if suitable membranes can be produced. Amphiphilic block copolymers (BCPs), developed as stable lipid analogs, form membranes that functionally incorporate membrane proteins and are ideal for such applications. While high protein density and planar membrane morphology are most desirable, BCP-membrane protein aggregates have so far been limited to low protein densities in either vesicular or bilayer morphologies. Here, we used dialysis to reproducibly form planar and vesicular BCP membranes with a high density of reconstituted aquaporin-0 (AQP0) water channels. We show that AQP0 retains its biological activity when incorporated at high density in BCP membranes, and that the morphology of the BCP-protein aggregates can be controlled by adjusting the amount of incorporated AQP0. We also show that BCPs can be used to form two-dimensional crystals of AQP0.

Aquaporin-0 membrane junctions form upon proteolytic cleavage.

Aquaporin-0 (AQP0), previously known as major intrinsic protein (MIP), is the only water pore protein expressed in lens fiber cells. AQP0 is highly specific to lens fiber cells and constitutes the most abundant intrinsic membrane protein in these cells. The protein is initially expressed as a full-length protein in young fiber cells in the lens cortex, but becomes increasingly cleaved in the lens core region. Reconstitution of AQP0 isolated from the core of sheep lenses containing a proportion of truncated protein, produced double-layered two-dimensional (2D) crystals, which displayed the same dimensions as the thin 11 nm lens fiber cell junctions, which are prominent in the lens core. In contrast reconstitution of full-length AQP0 isolated from the lens cortex reproducibly yielded single-layered 2D crystals. We present electron diffraction patterns and projection maps of both crystal types. We show that cleavage of the intracellular C terminus enhances the adhesive properties of the extracellular surface of AQP0, indicating a conformational change in the molecule. This change of function of AQP0 from a water pore in the cortex to an adhesion molecule in the lens core constitutes another manifestation of the gene sharing concept originally proposed on the basis of the dual function of crystallins.

Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility.

Selecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Nonconventional surfactants, such as amphipathic polymers (amphipols) and lipid bilayer nanodiscs, have been introduced as promising environments that may overcome intrinsic disadvantages of detergent micelle systems. However, structural insights into the effects of different environments on the embedded protein are limited. Here, we present a comparative study of the heptahelical membrane protein bacteriorhodopsin in detergent micelles, amphipols, and nanodiscs. Our results confirm that nonconventional environments can increase stability of functional bacteriorhodopsin, and demonstrate that well-folded heptahelical membrane proteins are, in principle, accessible by solution-NMR methods in amphipols and phospholipid nanodiscs. Our data distinguish regions of bacteriorhodopsin that mediate membrane/solvent contacts in the tested environments, whereas the protein's functional inner core remains almost unperturbed. The presented data allow comparing the investigated membrane mimetics in terms of NMR spectral quality and thermal stability required for structural studies.

Murine apolipoprotein serum amyloid A in solution forms a hexamer containing a central channel.

Serum amyloid A (SAA) is a small apolipoprotein that binds to high-density lipoproteins in the serum. Although SAA seems to play a role in host defense and lipid transport and metabolism, its specific functions have not been defined. Despite the growing implications that SAA plays a role in the pathology of various diseases, a high-resolution structure of SAA is lacking because of limited solubility in the high-density lipoprotein-free form. In this study, complementary methods including glutaraldehyde cross-linking, size-exclusion chromatography, and sedimentation-velocity analytical ultracentrifugation were used to show that murine SAA2.2 in aqueous solution exists in a monomer-hexamer equilibrium. Electron microscopy of hexameric SAA2.2 revealed that the subunits are arranged in a ring forming a putative central channel. Limited trypsin proteolysis and mass spectrometry analysis identified a significantly protease-resistant SAA2.2 region comprising residues 39-86. The isolated 39-86 SAA2.2 fragment did not hexamerize, suggesting that part of the N terminus is involved in SAA2.2 hexamer formation. Circular-dichroism spectrum deconvolution and secondary-structure prediction suggest that SAA2.2 contains approximately 50% of its residues in alpha-helical conformation and <10% in beta-structure. These findings are consistent with the recent discovery that human SAA1.1 forms a membrane channel and have important implications for understanding the 3D structure, multiple functions, and pathological roles of this highly conserved protein.