To disclose the interfacial behavior of proteins, in situ and in real-time degree information to which proteins maintain their original conformation at hydrophobic interfaces is critical. Based on energy landscapes theory, surface hydrophobicity and conformation flexibility in control (native) and pale, soft, and exudative-like (PSE-like (pre-denatured)) myosin at the hydrophobic interface was investigated. When the myosin concentration was lower than the keypoint concentration (Control: 0.05 mg/mL, PSE: 0.1 mg/mL), increasing myosin concentration improved the interfacial diffusion rate ( K diff ) and penetration rate ( K p ) at the oil/water interface. Although PSE myosin was easily adsorbed at the interface due to high surface hydrophobicity, hydrophobic aggregation and poor conformation flexibility led to low K diff and rearrangement rate ( K r ), respectively. According to the sum frequency generation (SFG) results, the proportion of the lost α-helix structures in PSE myosin (from 18.93% to 16.79%) was lower than that in control myosin (from 86.64% to 74.18%) after 3 h at the lipid/water surface in situ and in real-time. The difference in interfacial behaviors between control and PSE myosin may be rely on energy barriers from metastable PSE myosin and different unfolding pathways on energy landscapes between two proteins, supported by SFG. Macroscopically, confocal laser scanning microscopy and backscattering (BS) profiles results showed that unstable PSE myosin-soybean oil emulsion with poor interfacial behavior had a larger droplet size and a higher negative peak of ΔBS than control emulsion, respectively. These results have crucial implications for the regulation of interfacial behavior and emulsion characteristics in proteins with varied denatured states.
Two-dimensional (2D) lamellar membranes hold immense promise for sustainable separation technologies, yet their inherent dense interlayer architecture compromises permeability and selectivity. Herein, we propose a defect engineering strategy to construct hierarchical nanofluidic channels within graphene oxide (GO) membranes, by using intercalating polypyrrole (PPy) nanosheets with in-plane nanopores. The synergistic assembly of GO and PPy nanosheets enables tuning of both interlayer spacing and intra-plane nanopores, thereby generating multiscale transport pathways. The optimized PPy/GO membrane achieves a trade-off between selectivity and permeability: >96 % rejection for Congo red, <15 % rejection for salts (NaCl, Na 2 SO 4 , CaCl 2 ), and a remarkable water flux of 310 L m −2 h −1 bar −1 — 5-fold higher than state-of-the-art 2D membranes. Interface selective sum frequency generation (SFG) spectroscopy results reveal that PPy intercalation greatly enhances the order of hydrogen-bonded water at the membrane-water interface, which correlates with the fast and selective separation of dye and salt. Molecular dynamics and hydrodynamic simulations consolidate that the hierarchical channels enhance water permeation via defect-pore sieving, while selectively block organic dyes through size exclusion. Our work not only disentangles the intricate role of defect engineering in tailoring organic/salt separation for 2D membranes, but also provides a scalable approach for advanced separation systems in hypersaline wastewater treatment and resource recovery.
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