Vibrational sum frequency spectroscopy was used to probe fatty amine monolayers spread on various electrolyte solutions. The spectra revealed ion specific changes in both monolayer ordering and water structure with the former following the Hofmeister series. Separate measurements of the surface potential as a function of ion tracked closely to changes in alkyl chain structure, but less closely to changes in water structure. The disruption of the monolayer ordering could be ascribed to the relative ability of the ions to penetrate past the hydrophilic surface of the monolayer's headgroups and into the more hydrophobic portion of the thin film. The corresponding trends observed in the surface water structure showed significant deviations from the Hofmeister series, leading to the conclusion that the changes in surface water structure, often credited with being the origin of Hofmeister effects, are probably not of primary importance. On the other hand, dispersion forces almost certainly play a large role in the order of the Hofmeister series.

The molecular level details of the displacement of surface adsorbed fibrinogen from silica substrates were studied by atomic force microscopy, immunochemical assays, fluorescence microscopy, and vibrational sum frequency spectroscopy. The results showed that human plasma fibrinogen (HPF) can be readily displaced from the interface by other plasma proteins near neutral pH because the positively charged alpha C domains on HPF sit between the rest of the macromolecule and the underlying surface. The alpha C domains make weak electrostatic contact with the substrate, which is manifest by a high degree of alignment of Lys and Arg residues. Upon cycling through acidic pH, however, the alpha C domains are irreversibly removed from this position and the rest of the macromolecule is free to engage in stronger hydrogen bonding, van der Waals, and hydrophobic interactions with the surface. This results in a 170-fold decrease in the rate at which HPF can be displaced from the interface by other proteins in human plasma.

The behavior of poly(ethylene glycol) (PEG) conjugated lipids was investigated in planar supported egg phosphatidylcholine bilayers as a function of lipopolymer density, chain length of the PEG moiety, and type of alkyl chains on the PEG lipid. Fluorescence recovery after photobleaching measurements verified that dye-labeled lipids in the membrane as well as the lipopolymer itself maintained a substantial degree of fluidity under most conditions that were investigated. PEG densities exceeding the onset of the mushroom-to-brush phase transition were found to confer air stability to the supported membrane. On the other hand, substantial damage or complete delamination of the lipid bilayer was observed at lower polymer densities. The presence of PEG in the membrane did not substantially hinder the binding of streptavidin to biotinylated lipids present in the bilayer. Furthermore, above the onset of the transition into the brush phase, the protein binding properties of these membranes were found to be very resilient upon removal of the system from water, rigorous drying, and rehydration. These results indicate that supported phospholipid bilayers containing lipopolymers show promise as rugged sensor platforms for ligand-receptor binding.

We describe experiments and modeling results that reveal and explain the distribution of times that identical double-stranded DNA (dsDNA) molecules take to pass through a voltage-biased solid-state nanopore. We show that the observed spread in this distribution is caused by viscous-drag-induced velocity fluctuations that are correlated with the initial conformation of nanopore-captured molecules. This contribution exceeds that due to diffusional Brownian motion during the passage. Nevertheless, and somewhat counterintuitively, the diffusional Brownian motion determines the fundamental limitations of rapid DNA strand sequencing with a nanopore. We model both diffusional and conformational fluctuations in a Langevin description. It accounts well for passage time variations for DNA molecules of different lengths, and predicts conditions required for low-error-rate nanopore-strand DNA sequencing with nanopores.

Supported lipid bilayers (SLBs) have been widely used as model systems to study cell membrane processes because they preserve the same 2D membrane fluidity found in living cells. One of the most significant limitations of this platform, however, is its inability to incorporate mobile transmembrane species. It is often postulated that transmembrane proteins reconstituted in SLBs lose their mobility because of direct interactions between the protein and the underlying substrate. Herein, we demonstrate a highly mobile fraction for a transmembrane protein, annexin V. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the macromolecule but also passivate the underlying substrate to reduce nonspecific protein-substrate interactions. The thickness of the confined water layer can be tuned by fusing vesicles containing polyethyleneglycol (PEG)conjugated lipids of various molecular weights to a glass substrate that has first been passivated with a sacrificial layer of bovine serum albumin (BSA). The 2D fluidity of these systems was characterized by fluorescence recovery after photobleaching (FRAP) measurements. Uniform, mobile phospholipid bilayers with lipid diffusion coefficients of around 3 × 10 −8 cm 2 /s and percent mobile fractions of over 95% were obtained. Moreover, we obtained annexin V diffusion coefficients that were also around 3 × 10 −8 cm 2 /s with mobile fractions of up to 75%. This represents a significant improvement over bilayer platforms fabricated directly on glass or using single cushion strategies.

SNAREs such as VAMP, SNAP-25 and syntaxin are essential for intracellular trafficking, but what are their exact molecular roles and how are their interactions with other proteins manifest? Capitalizing on the differential sensitivity of SNAREs to exogenous proteases, we quantified the selective removal of identified SNAREs from native secretory vesicles without loss of fusion competence. Using previously established fusion assays and a high sensitivity immunoblotting protocol, we analyzed the relationship between these SNARE proteins and Ca2+-triggered membrane fusion. Neither the extent of fusion nor the number of intermembrane fusion complexes per vesicle were correlated with the measured density of identified egg cortical vesicle (CV) SNAREs. Without syntaxin, CVs remained fusion competent. Surprisingly, for one (but not another) protease the Ca2+dependence of fusion was correlated with CV SNARE density, suggesting a native protein complex that associates with SNAREs, the architecture of which ensures high Ca2+ sensitivity. As SNAREs may function during CV docking in vivo, and as further proteolysis after SNARE removal eventually ablates fusion, we hypothesize that the triggered steps of regulated fusion(Ca2+ sensitivity and the catalysis and execution of fusion)require additional proteins that function downstream of SNAREs.

TiO2 thin films coated on SiO2 substrates were prepared for the investigation of water structure at the TiO2/aqueous interface by vibrational sum-frequency spectroscopy (VSFS). The films were first characterized by X-ray photoelectron spectroscopy, ellipsometry, and atomic force microscopy. Film thickness could be readily varied between 0.9 and 5.9 nm with the thinnest film displaying the least degree of surface roughness. The 0.9 nm films were employed in the VSFS investigations and showed characteristic 3200 and 3400 cm -1 OH stretch frequencies of highly aligned interfacial water. Such peaks were reminiscent of those known from SiO2/aqueous interfaces; however, the chemistry at TiO2/aqueous interfaces was significantly richer. In the presence of Cl anions, the surface had an isoelectric point near pH 5.5 and showed the least degree of water organization near this pH. Almost equally strong 3200 cm -1 features could be produced at pH 2.0 and 12.0. On the other hand, the spectra were dramatically altered in the presence of phosphate-buffered saline. The phosphate ions specifically bound to the substrate surface and shifted the isoelectric point of the interface to pH 2.0. In this case, the intensity of the 3400 cm -1 peak was significantly increased in comparison with the Cl ion data at both neutral and acidic pH values. This is presumably because of a lack of sites directly adjacent to the oxide for forming tetrahedrally coordinated water when phosphate ions are present.

Difference in the vibes! The effect of divalent metal‐cation absorption on the structure of water at aqueous interfaces was explored in the presence of deprotonated fatty acid monolayers. Vibrational spectra were obtained from charged monolayers in the presence of Zn2+ and Mg2+ ions using vibrational sum frequency spectroscopy (see graphic). The differing modes of coordination by the two cations to the carboxylate headgroups led to significantly different water structures at the interfaces.