Recapitulating water wire geometry of AQP1 in OmpF mutants

Aquaporins have the ideal internal pore geometry for selective and highly permeable water channels but the pore wall interacts strongly with the permeating water wire47, indicating the possibility of enhancing permeability at similar size ranges without sacrificing selectivity. All aquaporins have a conserved asparagine-proline-alanine motif (known as the NPA motif) near the constriction region, in which the Asn interacts with the water wire by forming hydrogen bonds. These interactions impede the hydraulic permeability through AQP1. A recent study47 showed that in addition to the NPA motif there are 12 amino acids along the internal pore profile of AQP1 that can form hydrogen bonds with the water wire. Further, the number of hydrogen bonds between the water wire and the inner pore wall of AQPs was directly related to the single-channel permeability of the pore. Our aim is to redesign the water channel such that it minimizes interaction with the permeating water wire, thus eliminating hydrogen bonds in the central part of the channel but retaining the water wire geometry. To discern the unique water wire configurations (Supplementary Figure 1 and Supplementary Table 1) through AQP1, we examined individual frames of AQP1-water using all-atom 10 ns MD simulations (Fig. 1). Such water wires were subsequently positioned inside the OmpF pore and PoreDesigner was used to alter the pore-constricting residues to form the equivalent of a molecular mold around the water wire (Fig. 1).

Categorization of redesigned pores

Water wires were placed inside the WT OmpF (2omf.pdb) pore and used as input for PoreDesigner. The pore-constricting residues were altered to fill up the annular space using hydrophobic, long side-chain amino acids with the objective of designing a narrow yet hydrophobic pore with minimal water−wire interaction. An explicit constraint was imposed to ensure that the distance between any hetero-atom of the pore constriction residue and the water wire oxygen was greater than the sum of their van der Waals radii to preclude the possibility of arriving at designs that occlude the pore. PoreDesigner reduces binding with the central water wire by maintaining their respective interaction energies at their maximum by replacing the pore constriction residues of WT OmpF with long side-chain, hydrophobic amino acids such as tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr).

PoreDesigner yielded 40 different OmpF designs with pore sizes less than 4 Å. Analysis of the engineered designs revealed that they conform to three categories (Fig. 1) based on the resultant internal pore geometry: (1) only Trp/Phe mutations resulting in a narrower but off-center pore lumen (OCD: off-center pore closure design), (2) a smaller cocentric pore with the bulky groups (such as Trp, Phe, and Tyr) interspersed with less bulky alanines and valines arranged in a single plane (UCD: uniform pore closure design), and (3) regularly patterned larger with smaller side-groups along the pore profile resulting in an internal pore geometry that involves a twist (cork-screw designs—CSDs). There were two off-center (OCD) designs for which we allowed the mutation of the 25 pore constriction residues to: (a) only Phe mutations (TFPhe), and (b) only Trp mutations (TFTrp). A biased distribution of bulky groups (Phe/Trps) towards one side of the pore periphery resulted in a smaller pore with its center away from the large residues. The presence of 25 Phe/Trps led to steric clashes forcing most of the Phe/Trps side chains to face away from the pore lumen (Fig. 2) thus enhancing pore wall hydrophobicity only. In the remaining 38 designs, all hydrophobic long side-chain amino acids were permitted, thus enabling them to sample smaller pore sizes by placing long side-chain residues interspersed with short amino acids (generally alanines, valines, and leucines). These designs (UCD) alleviate the possibility of a Phe-Phe/Trp-Trp side-chain steric clashes akin to the OCD designs (Fig. 1). We identified 31 UCD and seven CSD designs. We chose the smallest predicted pore size design from each type for subsequent osmotic permeability and solute rejection experiments and molecular dynamics simulations. The predicted pore constriction dimensions after MD simulations for the three selected designs were: 3.54 × 3.25 Å, 3.18 × 3.12 Å, and 3.05 × 3.01 Å for OCD, CSD, and UCD protein designs, respectively. These OmpF mutant proteins and the WT OmpF proteins were produced by expression from synthetic genes cloned into the pET23a(+) expression plasmid vector transformed into E. coli BL21(DE3) Omp8 Rosetta (ΔlamBompF::Tn5 ΔompAΔompC) mutant strain. The purified proteins were incorporated into liposomes for assessment of single-channel water permeability and solute passage as described in subsequent sections.

Fig. 2 OCD-TFTrp design shows steric clash but UCD does not. The OCD_TFTrp design has adjacent tryptophans clashing (left) resulting in some of the side chains facing away from the pore lumen, thereby yielding pore sizes larger than expected. However, in a UCD design (right), an R82L mutation alleviates a steric clash with Trp62 (unlike OCD-TFTrp). UCD designs are seen to intersperse smaller side-chain hydrophobic amino acids between longer ones so their side chains face the pore lumen resulting in smaller pore sizes Full size image

Before testing the three designs for water transport, the Kyte−Doolittle hydrophobicity scores of the three designs were calculated (Supplementary Methods) and contrasted with that of WT OmpF (Table 1). The relative order of inner pore wall hydrophobicities was seen to be CSD > OCD > UCD > wild type OmpF. We hypothesized that the experimentally measured single-channel permeabilities will follow the same trend as increasing hydrophobicity based on our design principle of eliminating water−pore wall interactions to enhance permeability.

Table 1 Ranges of values for the inner pore wall, outer pore wall, and overall hydrophobicity scores Full size table

The hydrophobicity trends reveal that the OCD-TFTrp mutant has the highest estimated outer pore wall hydrophobicity. This is possibly due to the steric clashes between contiguous tryptophans (Fig. 2) where the majority of the 25 pore-constricting tryptophans are forced away from the lumen.

In addition, we also used PoreDesigner to predict designs that span the remaining 4–10 Å range. The overall goal is to precisely match any desired pore size needed for separations spanning the sub-nm (3–10 Å) range. PoreDesigner was accordingly modified to only accept pore designs with pore constriction diameter between a prespecified range Dmin and Dmax. For example, setting Dmin and Dmax values to 5 and 6 Å respectively yields OmpF designs with pores predicted to be within this range. We identified 17 additional designs (Fig. 3) with at least two designs within a pore size bin of range 1 Å starting from 4 to 9 Å. We used the aforementioned structural classification scheme and developed ten OCD designs, three UCD and four CSD designs. Generally, the smaller the desired pore size, the higher was the number of required mutations. OCD type designs were seen to be most prevalent spanning almost the entire sub-nm range (Fig. 3b). Whereas CSD and UCD type designs were limited to the mid region of the sub-nm spectrum.

Fig. 3 Twenty OmpF mutants spanning the entire sub-nm range were designed. a Plot of the number of mutations vs. pore diameter for 20 mutants (including three mutants that were validated experimentally before MD simulations). The general trend indicates that the smaller the desired pore, the greater the number of mutations required. b Plot of the number of designs for each pore size and type classification Full size image

Experimental validation of pore designs

WT OmpF and three OmpF mutants were chosen to measure their single-channel permeability and solute passage rates experimentally. The three OmpF mutants that we chose were OCD with in silico estimated pore sizes of 3.25 Å (minor axis of elliptical pore cross-section), CSD with pore size of 3.12 Å, and UCD with estimated pore size of 3.01 Å. The amino acid sequences of these mutant proteins are provided in Supplementary Figure 6.

Solute rejection of OmpF mutants

We estimated the approximate molecular weight limit at which solute rejection for WT OmpF and the three OmpF mutants occurred using stopped-flow light scattering experiments (see Fig. 4 for details). For WT OmpF, the light scattering intensity decreased at the second stage when WT OmpF-reconstituted liposomes were exposed to NaCl containing hypertonic solutions. Also, the light scattering intensity decreased at the second stage when WT OmpF-reconstituted liposomes were exposed to glycine, glucose or sucrose containing hypertonic solutions. This indicates that WT OmpF is permeable to these solutes. The light scattering intensity leveled off when exposing the proteoliposomes to PEG600 containing hypertonic solutions (Fig. 4). This observation demonstrated that WT OmpF can reject PEG600 (600 Da) or larger molecules, which is consistent with previous reports35.

Fig. 4 Osmotic shock stopped-flow light scattering experiments were used to assess transport properties. Stopped-flow light scattering experiments revealed an order of magnitude or higher permeability than aquaporins for WT OmpF protein and its mutants as well as solute retention trends seen in OmpF protein mutants. a When OmpF (or OmpF mutant) containing proteoliposomes are mixed with hypertonic solutions, two different transport models can be observed based on whether the solute is permeable to the porin or not. b In the stopped-flow setup, for solute excluded model, normalized light scattering intensity levels off during the second stage as there is no inflow of water and solutes; for solute permeable model, normalized light scattering intensity decreased during the second stage due to inflow of water and solutes. c OmpF (WT) rejects PEG600 (600 Da) and larger molecules and thus only the PEG600 curves show no decreasing portion of the curve. d UCD rejects NaCl (58.5 Da) and larger molecules as there is no decreasing portion of the stopped-flow curve for any of the solutes tested. e Summary of the estimated solute rejection (light bars) and single-channel permeability (dark bars) of OmpF WT and the three OmpF mutants (details in Supplementary Figures 3 and 4). The two y-axes represent permeability (black left y-axis) or the molecular weight cutoff data (red right y-axis). Curves shown in panels c and d are averages of 6–10 traces from each stopped-flow light scattering experiment. Each experiment was conducted at least three times with independent vesicle preparations (complete data in Supplementary Methods and Supplementary Figure 3) Full size image

For UCD, the light scattering intensity leveled off when exposing the liposomes to all the solutes used including NaCl (58.5 Da), leading us to conclude that this mutant can substantially reject molecules larger than 58.5 Da (Fig. 4). We also estimated the approximate molecular weight exclusion limit for OCD and CSD (Supplementary Methods and Supplementary Figure 3 for details). Based on the solute rejection experiments above, we estimated the molecular exclusion limit of the three mutants to have the following sequence: WT (~600 Da) > OCD (~342 Da) > CSD (~180 Da) > UCD (~58 Da) (Fig. 4), which follows the same trend as the designed pore sizes. Thus, small molecule separation membranes can be developed by a selection of different pore size OmpF mutants for biomimetic membranes. The mutant with the smallest pore size, UCD, has ionic solute rejection properties similar to aquaporins (while not excluding protons), and can be selected as a candidate protein for developing membrane protein-based biomimetic desalination membranes.

Single-channel permeability of OmpF mutants

Recent literature has focused on emphasizing the importance of membrane design efforts that lead to high selectivity while maintaining or increasing current membrane permeabilities15. Solute rejection experiment results showed that high molecular selectivity can be achieved by designing OmpF mutants with different pore sizes through the PoreDesigner workflow. In addition to estimating selectivity, we also evaluated OmpF mutant permeabilities, which were specifically characterized by determining single-channel permeability of each mutant.

Figure 4 shows light scattering curves obtained from vesicle membranes with reconstituted WT and mutant OmpF proteins at a lipid to protein mass ratio of 400 (LPR400), with net permeabilities between 2049 and 3411 μm/s. Because net permeability can depend both on the number of proteins reconstituted per vesicle and the single-channel permeability29, for more accurate comparison between mutants, we calculated the single-channel permeability of WT OmpF and its mutants. By taking the ratio of the number of proteins to the number of vesicles, we can obtain the average number of proteins per vesicle (N pro /N ves ) (Supplementary Figure 5). Combining vesicle permeability and average number of OmpF proteins per vesicle, we calculated average single-channel permeability of OmpF and its mutants (Fig. 4). CSD had the highest single-channel permeability followed by OCD and UCD, which have similar single-channel permeabilities and all the three mutants have single-channel permeabilities higher than WT OmpF. This serves as the experimental corroboration of the predicted water permeation rates from the inner pore wall hydrophobicities. Compared to aquaporins, single-channel permeabilities of WT OmpF and its mutants are at least an order of magnitude higher48, the highest single-channel water permeability of OmpF mutants is ~18 times faster than that of the E. coli AqpZ, which was measured using the same platform29 and ~49 times faster than that of AQP1 reported in literature45.

Molecular dynamics simulation of the pore designs

Using the all-atom MD method, we independently assessed osmotic permeability of the WT OmpF and the three experimentally verified designs, starting from the molecular configurations suggested by PoreDesigner. The monomeric proteins were patched to form trimers using a VMD49 plugin, set in a lipid-bilayer and solvated in 1 M NaCl solution (Fig. 5). Osmotic permeabilities were evaluated (from the rate of vesicle volume changes50) and averaged through each monomer of the trimeric molecule and the variabilities during the last 30 ns of the 35 ns simulation were reported (Fig. 5). The MD-computed osmotic permeabilities of OmpF and the three designs are seen to corroborate the same single-channel permeability trend as seen in stopped-flow light scattering experiments. Highest permeability of CSD reaffirms the applicability of using inner pore wall hydrophobicity scores as a surrogate to predict relative channel permeabilities.

Fig. 5 MD simulations of OmpF corroborate experimentally observed permeability and selectivity trends. a Typical simulation system. (Top) Cut-away view of the system revealing a transmembrane water passage through an OmpF monomer. The OmpF monomer is depicted in purple, the lipid-bilayer in cyan, water molecules as red and white spheres, and Na+ and Cl– ions as orange and green spheres, receptively. (Bottom) Top-down view of the system. The OmpF trimer is drawn using a cartoon representation, the lipid-bilayer as cyan bonds; water and ions are not shown. b Simulated osmotic permeability (averaged over 12,500 frames) of OmpF variants (red) and the corresponding experimental values (gray). c Ionic conductance of OmpF trimers obtained from applied field simulations under a 500 mV transmembrane voltage and averaged over 10,417 frames. d Water occupancy of OmpF variants. The green volume depicts the average location of water molecules in each channel characterized as a 0.3 g/cm3 isosurface of water oxygen density. For reference, each channel is shown using a semitransparent cartoon representation. e Major axis dimensions of the pores measured from PoreDesigner before MD (gray) and from the last 100 frames of MD (red). The error bars represent standard deviations. A 0.4 nm line represents the PoreDesigner design constraint of identifying pore designs smaller than 0.4 nm. f The average number of hydrogen bonds made between water and an OmpF monomer in each of the regions depicted in panel d and averaged over 14,583 frames Full size image

The ionic conductances of the WT and mutant pores were evaluated by simulating the systems under a transmembrane voltage of 500 mV (Fig. 5). The ionic conductance was determined by averaging instantaneous displacements of ions over the last 25 ns of the 35 ns MD trajectoires51. All the three mutants exhibited negligibly low conductances which were about an order of magnitude lower than that of WT OmpF with the CSD mutant being the most conductive of the three as expected from solute rejection experiments. While solute rejection experiments identified UCD to be more restrictive towards salt compared to OCD, MD simulations, as conducted, do not seem to be conclusive regarding the difference between the two mutants (Fig. 5).

Further analysis of the MD trajectories identified the inner volume of the WT and mutant pores accessible to water (Fig. 5). The volumes were determined by averaging water density over the last 10 ns of the MD simulations carried out under a 500 mV bias. The volume density maps reveal the UCD mutant to have the narrowest pore constriction, which correlates with the best solute rejection performance of that mutant. Figure 5 shows the major pore constriction diameters as measured using PoreDesigner (before MD simulations were performed) along with that observed during the 5 ns of MD. The closely packed side chains of OCD and UCD allowed marginal movement of pore constriction amino acid side chains, thereby showing less variability in the pore size during the course of MD. However, the bulky groups of CSD are stacked in different planes thus allowing some movement of the pore constriction side chains leading to higher variability in pore sizes during water permeation. The same PoreAnalyzer module (Supplementary Methods) that was used in PoreDesigner was used for assessing the pores from the MD trajectories.

To assess the effect of relative hydrophobicity of the mutant pores, we determined the average number of hydrogen bonds between the OmpF monomer and water located in each of the three regions of the pores (defined in Fig. 5). A hydrogen bond was reported if the water molecule was within 0.3 nm of a protein atom capable of forming a hydrogen bond similar to as explained in Ireta et al.52 and Durrant et al.53. It was also ensured that the protein atom–water hydrogen–water oxygen angle was 20° or less. The number of calculated bonds was averaged over entire 35 ns MD trajectories with error bars showing standard deviations (Fig. 5). The constriction region (Fig. 5) shows progressively decreasing number of hydrogen bonds as the number of mutations (in the constriction region) as the pore is occluded by more hydrophobic residues. This attests to the effectiveness of PoreDesigner’s design objective of replacing pore constriction residues with hydrophobic ones to limit pore wall−water wire interactions in order to arrive at designs with high single-channel permeabilities while tuning size selectivity.