(169d) Optimizing the Membrane Composition Surrounding E. coli Amtb Ammonium Transporter Proteins for Highly Ammonium-Selective Protein-Loaded Biomimetic Membranes

Recovering valuable products from wastewater can reduce energy inputs, costs, and environmental damage associated with pollution mitigation and chemical manufacturing but requires highly selective separation materials integrated into effective unit processes. The advantages of selective resource recovery materials are especially apparent within the nitrogen cycle. Roughly 1% of global energy is used by the Haber-Bosch process to convert atmospheric dinitrogen gas (N₂) to ammonia (NH₃), producing CO₂ as a byproduct. Anthropogenic sources of reactive nitrogen have unbalanced the nitrogen cycle because 80% of wastewater globally is discharged without treatment, and reactive nitrogen threatens aquatic ecosystems with eutrophication. Moreover, conventional biological wastewater treatment converts 100-fold less reactive nitrogen back to N₂ per year than is generated by Haber-Bosch, necessitating additional techniques that supplement biological treatment. Materials capable of selectively recovering reactive nitrogen from wastewaters without chemical transformation can provide an economic incentive for expanding global environmental remediation and can mitigate dependence on both the Haber-Bosch process and biological wastewater treatment.

In biology, membrane transporter proteins are responsible for moving atoms and molecules through cell membranes with an extremely high degree of selectivity. Therefore, these proteins are attractive options for incorporation into synthetic membranes that also aim to be highly selective towards one or a few targets. The most well-studied example is aquaporins, selective and highly permeable water transport proteins, and biomimetic aquaporin-embedded membranes have already been commercialized for water desalination. However, a major challenge is ensuring that the bio-membrane layer shows good stability, holds the maximum concentration of proteins for high permeability, and preserves correct protein folding. The design of such a layer may be unique for each protein according to each protein’s binding characteristics.

E. coli AmtB ammonium transporter protein (EcAmtB) is a promising candidate for an ammonium-selective biomimetic membrane because its crystal structure and transport mechanism have been extensively characterized, and it has proven to be selective for ammonium outside of a living cell (K_d,Na+ / K_d,NH4+ of roughly 1700). However, it has also recently been reported that EcAmtB does not transport ions well in the absence of POPG, a glycerol-terminated phospholipid found in E. coli native membranes. Reports have investigated multiple binding domains for both POPG and POPE, an ethanolamine-terminated phospholipid also native to E. coli. Both seem to greatly stabilize the protein and cause correct folding into an effective transporter. A difficulty of this reliance is that phospholipids are often considered to be less stable in synthetic membranes compared to synthetic copolymers. Hence, it is imperative that the membrane composition dependence of EcAmtB is extensively investigated before a final membrane is constructed.

To this end, solid supported membrane-based electrophysiology (SSME) is used to analyze ammonium permeability and selectivity in multiple proteoliposomes containing EcAmtB and various membrane compositions, including both phospholipids and synthetic copolymers. Preliminary results with proteoliposomes containing binary phospholipid compositions POPE/POPG, POPC/POPG, POPE/PA, and POPC/PA suggest that POPE may be an essential phospholipid for EcAmtB. The replacement of POPG with phosphatidic acid (PA) resulted in a marginal worsening in K_m, but the replacement of POPE with the choline-terminated POPC resulted in negligible ammonium transport at initial ammonium concentrations from 1 mM to 50 mM.

Ultimately, transporter protein-loaded biomimetic membranes can potentially aid in advancing a future vision of a circular nitrogen economy that extracts ammonium pollutants from wastewaters and offsets anthropogenic imbalances to the global nitrogen cycle.