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pH-sensitive viral fusion protein mimics are widely touted as a promising route towards site-specific delivery of therapeutic compounds across lipid membranes. Here, we demonstrate that a fusion protein mimic, designed to achieve a reversible, pH-driven helix-coil transition mechanism, retains its functionality when covalently bound to a surface.

GALA is a 30 amino acid synthetic peptide consisting of a Glu-Ala-Leu-Ala repeat and is known to undergo a reversible structural transition from a disordered to an α-helical structure when changing the pH from basic to acidic values. In its helical state GALA can insert into and disintegrate lipid membranes. This effect has generated much interest in GALA as a candidate for pH triggered, targeted drug delivery. GALA also serves as a well-defined model system to understand cell penetration mechanisms and protein folding triggered by external stimuli. Structural transitions of GALA in solution have been studied extensively. However, cell penetration is an interfacial effect and potential biomedical applications of GALA would involve a variety of surfaces, e.g., nanoparticles, lipid membranes, tubing, and liquid-gas interfaces. Despite the apparent importance of interfaces in the functioning of GALA, the effect of surfaces on the reversible folding of GALA has not yet been studied. Here, we use sum frequency generation vibrational spectroscopy (SFG) to probe the structural response of GALA at the air-water interface and IR spectroscopy to follow GALA folding in bulk solution. We combine the SFG data with molecular dynamics simulations to obtain a molecular-level picture of the interaction of GALA with the air-water interface. Surprisingly, while the fully reversible structural transition was observed in solution, at the water-air interface, a large fraction of the GALA population remained helical at high pH. This "stickiness" of the air-water interface can be explained by the stabilizing interactions of hydrophobic leucine and alanine side chains with the water surface.

We describe a microscope for measuring two-dimensional infrared (2D IR) spectra of heterogeneous samples with μm-scale spatial resolution, sub-picosecond time resolution, and the molecular structure information of 2D IR, enabling the measurement of vibrational dynamics through correlations in frequency, time, and space. The setup is based on a fully collinear "one beam" geometry in which all pulses propagate along the same optics. Polarization, chopping, and phase cycling are used to isolate the 2D IR signals of interest. In addition, we demonstrate the use of vibrational lifetime as a contrast agent for imaging microscopic variations in molecular environments.

Lipid storage in plants is achieved among all plant species by formation of oleosomes, enclosing oil (triacylglycerides) in small subcellular droplets. Seeds are rich in this pre-emulsified oil to provide a sufficient energy reservoir for growing. The triacylglyceride core of the oleosomes is surrounded by a phospholipid monolayer containing densely packed proteins called oleosins. They are anchored in the triacylglycerides core with a hydrophobic domain, while the hydrophilic termini remain on the surface. These specialized proteins are expressed during seed development and maturation. Particularly, they play a major role in the stabilization and function of oleosomes. To better understand the importance of oleosins for oleosome stabilization, enzymatic digestion of oleosins was performed. This made it possible to compare and correlate changes in the molecular structure of oleosins and changing macroscopic properties of oleosomes. Tryptic digestion cleaves the hydrophilic part of the oleosins, which is accompanied by a loss of secondary structures as evidenced by Fourier-transform infrared and sum frequency generation spectra. After digestion, the ability of oleosins to stabilize oil-water or air-water interfaces was lost. The surface charge and the associated aggregation behavior of oleosomes are governed by interactions typical of proteins before digestion and by interactions typical of phospholipids after digestion.