Membrane Protein Insertion into and Compatibility with Biomimetic Membranes

Abstract

Membrane protein and membrane protein–mimic functionalized materials are rapidly gaining interest across a wide range of applications, including drug screening, DNA sequencing, drug delivery, sensors, water desalination, and bioelectronics. In these applications, material performance is highly dependent on activity‐per‐protein and protein packing density in bilayer and bilayer‐like structures collectively known as biomimetic membranes. However, a clear understanding of, and accurate tools to study these properties of biomimetic membranes does not exist. This paper presents methods to evaluate membrane protein compatibility with biomimetic membrane materials. The methods utilized provide average single protein activity, and for the first time, provide experimentally quantifiable measures of the chemical and physical compatibility between proteins (and their mimics) and membrane materials. Water transport proteins, rhodopsins, and artificial water channels are reconstituted into the full range of current biomimetic membrane matrices to evaluate the proposed platform. Compatibility measurement results show that both biological and artificial water channels tested largely preserve their single protein water transport rates in biomimetic membranes, while their reconstitution density is variable, leading to different overall membrane permeabilities. It is also shown that membrane protein insertion efficiency inversely correlates with both chemical and physical hydrophobicity mismatch between membrane protein and the membrane matrix.

PoreDesigner for tuning solute selectivity in a robust and highly permeable outer membrane pore

“…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 higher 48 , 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 platform 29 and ~49 times faster than that of AQP1 reported in literature 45 .…”

“…The permeabilities of tested designed ranged from 1.0 (±0.23)×10 −12 cm 3 /s for the WT to 4.4 (±0.93)×10 −12 cm 3 /s (the ±values are standard deviations based on measurements from three replicates in both WT and CSD) for the selected CSD design compared to ~9×10 −14 cm 3 /s estimated for AQP1 45 . CSD osmotic water permeabilities were not only higher than AQP1 by an order of magnitude but were also an order of magnitude higher than the highest reported permeabilities of any channel of the aquaporin family of proteins (2.4 (±0.47)×10 −13 cm 3 /s determined for Rhodobacter sphaeroides AqpZ 29 , 46 ). Solute rejection capabilities of these channels were evaluated using stopped-flow experiments with various solutes.…”

“…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 permeability 29 , 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 ).…”

“…We characterized the influx of different molecular weight solutes through WT OmpF and its mutants under hypertonic conditions using stopped-flow light scattering measurements and compared their solute transport trends. All OmpF mutants were reconstituted into l -α-phosphatidylcholine (PC)/ l -α-phosphatidylserine (PS) vesicles using a detergent destabilization method at LPR400 29 . Briefly, vesicles were created by rehydrating dry lipid films in rehydration buffer (20 mM HEPES, 100 mM NaCl, 0.02% (wt/v) NaN 3 , pH7.4) and then extruded through 200 nm track-etched membranes.…”

“…Rapid and selective water permeation through AQPs at ~3 billion water molecules/channel/s, makes them ideal for desalination. However, the advantages of AQPs for high permeability and high selectivity membrane applications are still being debated due to questions regarding the long-term stability of this alpha helical protein 28 , the low density of proteins embedded in membranes 29 , and its pore wall chemistry which forms hydrogen bonds with the permeating water 30 molecules that may impede single-channel osmotic permeability. Further, AQP-based membranes do not allow for selective removal of larger solutes in the sub-nm range.…”