Organization of AQP0 Tetramers in the Different 2D Crystal Forms.

<p><b>A</b> In 2D crystals formed with DMPG that show <i>p</i>42₁2 symmetry, a tetramer in one membrane sits in the center of four tetramers in the opposing membrane, thus making contact with subunits of four separate tetramers. <b>B</b> In crystals with <i>p</i>12₁ symmetry, the tetramer in one membrane is shifted by half a unit cell length (32.75 Å) in one lattice direction but by less than half a unit cell length in the other lattice direction (28.7 Å). As in the <i>p</i>42₁2 crystals, each tetramer makes contact with subunits of four separate tetramers in the opposing membrane. <b>C</b> AQP0 tetramers in the two membranes in 2D crystals formed with DMPC are exactly in register, establishing <i>p</i>422 symmetry, and each tetramer thus only interacts with subunits of a single tetramer in the opposing membrane. 2D crystals formed with DMPG that show <i>p</i>422 symmetry likely have the same tetramer arrangement as the one shown here for 2D crystals formed with DMPC. The lower panels show the distances between the two layers in the different crystal forms.</p

Representative Images of Negatively Stained Samples of AQP0 Reconstituted with the Anionic Lipids DMPS and DMPA.

<p><b>A</b> AQP0 reconstituted with DMPS at pH 6.0 yielded 2D crystals that showed a pronounced tendency to stack (left panel). Incubation of these crystals with pH 8 buffer resulted in partial unstacking of the crystals (middle panel), and incubation with pH 10 buffer completely separated the crystals (right panel). All crystals under all pH conditions always showed <i>p</i>422 symmetry (inset in left panel). <b>B</b> Reconstitution of AQP0 with DMPA at pH 6.0 resulted in protein aggregates and presumably mostly empty vesicles (left panel). Only DMPE/DMPA mixtures that contained 20% or 40% DMPA (w/w) (middle and right panel, respectively) resulted in the formation of 2D crystals, which were always of <i>p</i>422 symmetry (insets). Scale bars are 2 μm.</p

Percentage of AQP0 2D Crystals with a Given Symmetry Formed at Different DMPG:DMPE Ratios.

<p>Up to a DMPG concentration of 40% (w/w), all crystals showed <i>p</i>422 symmetry. At higher DMPG concentrations, the percentage of crystals with <i>p</i>422 symmetry decreased as increasingly more crystals with other symmetries formed. The results shown are averages of three independent experiments, and the bars represent standard deviation. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117371#pone.0117371.s005" target="_blank">S2 Table</a>.</p

Electron crystallographic statistics for the 3D reconstructions obtained with AQP0 2D crystals grown with DMPG with <i>p</i>42₁2 symmetry (AQP0_DMPG-p4212) and <i>p</i>12₁ symmetry (AQP0_DMPG-p121).

<p>Electron crystallographic statistics for the 3D reconstructions obtained with AQP0 2D crystals grown with DMPG with <i>p</i>42₁2 symmetry (AQP0_DMPG-p4212) and <i>p</i>12₁ symmetry (AQP0_DMPG-p121).</p

Double-Layered 2D Crystals of AQP0 Grown with DMPG.

<p><b>A</b> Image of a representative AQP0 2D crystal formed with DMPG in negative stain. Scale bar is 1 μm. <b>B</b> High magnification view of an edge of the crystal. The white arrows mark the two parallel edges of the double-layered crystal. Scale bar is 50 nm. <b>C</b> Contour representations of four projection maps at ~7 Å resolution calculated from images of AQP0 2D crystals with <i>p</i>422, <i>p</i>42₁2, <i>p</i>12₁, and <i>p</i>1 symmetry (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117371#pone.0117371.s004" target="_blank">S1 Table</a>). All crystal forms have the same unit cell of <i>a</i> = 65.5 Å, <i>b</i> = 65.5 Å, and γ = 90.0°. The solid and dashed squares indicate the relative position of AQP0 tetramers in the two crystalline layers of the double-layered crystals. For projection maps other than that of the crystal with <i>p</i>422 symmetry, the locations of the tetramers were determined by cross correlation with the <i>p</i>422 projection map. The different crystal symmetries result from shifts between the two crystalline layers in the direction parallel to the lattice vectors.</p

Docking of the Atomic Model of AQP0 into the 3D Density Maps of the DMPG Crystals with <i>p</i>42₁2 and <i>p</i>12₁ Symmetry.

<p><b>A</b> Density map at 7 Å resolution calculated from images of AQP0 2D crystals with <i>p</i>42₁2 symmetry (purple) with the docked atomic model (blue). <b>B</b> Density map at 8 Å resolution calculated from images of AQP0 2D crystals with <i>p</i>12₁ symmetry before non-crystallographic symmetry (NCS) averaging. <b>C</b> The map calculated from images of AQP0 2D crystals with <i>p</i>12₁ symmetry was improved by NCS averaging (green). The docked atomic model of AQP0 is shown in blue.</p

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

A Prodomain Fragment from the Proteolytic Activation of Growth Differentiation Factor 11 Remains Associated with the Mature Growth Factor and Keeps It Soluble

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor β (TGF-β) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60–114, PDP_60–114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP_60–114 had no impact on activity. The specific activity of the GDF11/PDP_60–114 complex (EC₅₀ = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP_60–114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP_60–114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP_60–114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC₅₀ = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC₅₀ = 2 nM) by protease treatment. Complex formation with PDP_60–114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-β family that form latent pro/mature domain complexes