The increasing demand for freshwater for food production, processing and energy generation has made the efficient utilization of marginal water resources an imperative global environmental objective. Ultra-permeable membranes (dense solution, diffusion-based or channel-based) have emerged as key industrial workhorses for the same. Aquaporins (AQPs) have emerged as ideal candidates for channel-based membranes owing to their ~billion molecules/sec hydraulic permeabilities. Outer membrane porin type-F (OmpF) are beta-barrel proteins have also found relevance in channel-based membranes employed for aqueous-phase separations due to their ability to survive contacts with 100% ethanol. To this end, we systematically redesigned the pore constricting residues of OmpF using a modified version of Iterative Protein Redesign & Optimization (IPRO) suite (1) of programs to perturb the OmpF protein backbone, mutate and recursively repack the sidechains in order to obtain the selective internal structure of AQP (pore constriction diameter ~4 times smaller than OmpF) inside the stable beta-scaffold of OmpF (pore constriction diameter ~1.1nm). We first generated an initial set of designs and performed MD simulations and subsequent experiments using stopped-flow light scattering experiments to validate three designs, which are unique in their internal pore geometries. We subsequently generated 17 more designs with a modified objective of sampling pore sizes across the 0.2-1 nm range. The primary objective of this work is to redesign OmpF with any user-defined pore size without compromising on the stability of the mutated protein and thereafter assess its transport properties such as, hydraulic permeability and solute rejection.
Pore sizes between 0.3 nm and 0.5 nm are key to several industrial and environmental separations such as methane/CO2 and salt/water, which is accessible to the less stable AQP1. We classified the IPRO designs into three broad classes based on the final pore geometry. A cork-screw (CSD) class of IPRO-suggested design (pore constriction ~0.236 nm by ~0.194 nm) shows alternately stacked bulky groups along the pore periphery, which when expressed, purified and immobilized on a porous support, exhibits high hydraulic permeability and not only completely rejects glycine (75 Da) but also shows ~70% salt (58 Da) rejection. Another type of mutant (with 25 Trp mutations) shows a highly hydrophobic pore (pore constriction ~0.33 nm by ~0.35 nm) that exhibits several folds higher hydraulic permeabilities than AQP1. Herein we propose a platform for designing precisely tuned sub-nm membrane transporters which will play a pivotal role both in performing energy-efficient water treatment and vesicle mediated highly-selective transport of antibiotics. Subsequent efforts will be directed towards systematic mutation of membrane-facing residues for the OmpF mutant series of proteins to tune interactions with various biomimetic membrane materials.
1. Pantazes RJ, Grisewood MJ, Li T, Gifford NP, Maranas CD. The Iterative Protein Redesign and Optimization (IPRO) suite of programs. J Comput Chem. 2015;36(4):251-63.