Windowless detection geometry for sum frequency scattering spectroscopy in the C-D and amide I regions.

Understanding the structure and chemistry of nanoscopic surfaces is an important challenge for biointerface sciences. Sum frequency scattering (SFS) spectroscopy can specifically probe the surfaces of nanoparticles, vesicles, liposomes, and other materials relevant to biomaterial research, and, as a vibrational spectroscopy method, it can provide molecular level information about the surface chemistry. SFS is particularly promising to probe the structure of proteins, and other biological molecules, at nanoparticle surfaces. Here, amide I spectra can provide information about protein folding and orientation, while spectra in the C-D and C-H stretching regions allow experiments to determine the mode of interaction between particle surfaces and proteins. Methods used currently employ a closed liquid cell or cuvette, which works extremely well for C-H and phosphate regions but is often impeded in the amide I and C-D regions by a strong background signal that originates from the window material of the sample cells. Here, we discuss a windowless geometry for collecting background-free and high-fidelity SFS spectra in the amide I and C-D regions. We demonstrate the improvement in spectra quality by comparing SFS spectra of unextruded, multilamellar vesicles in a sample cuvette with those recorded using the windowless geometry. The sample geometry we propose will enable new experiments using SFS as a probe for protein-particle interactions.

Synthetic Artificial Apoptosis-Inducing Receptor for On-Demand Deactivation of Engineered Cells.

The design of a fully synthetic, chemical "apoptosis-inducing receptor" (AIR) molecule is reported that is anchored into the lipid bilayer of cells, is activated by the incoming biological input, and responds with the release of a secondary messenger-a highly potent toxin for cell killing. The AIR molecule has four elements, namely, an exofacial trigger group, a bilayer anchor, a toxin as a secondary messenger, and a self-immolative scaffold as a mechanism for signal transduction. Receptor installation into cells is established via a robust protocol with minimal cell handling. The synthetic receptor remains dormant in the engineered cells, but is effectively triggered externally by the addition of an activating biomolecule (enzyme) or in a mixed cell population through interaction with the surrounding cells. In 3D cell culture (spheroids), receptor activation is accessible for at least 5 days, which compares favorably with other state of the art receptor designs.

Orientation and Conformation of Proteins at the Air-Water Interface Determined from Integrative Molecular Dynamics Simulations and Sum Frequency Generation Spectroscopy.

Understanding the assembly of proteins at the air-water interface (AWI) informs the formation of protein films, emulsion properties, and protein aggregation. Determination of protein conformation and orientation at an interface is difficult to resolve with a single experimental or simulation technique alone. To date, the interfacial structure of even one of the most widely studied proteins, lysozyme, at the AWI remains unresolved. In this study, molecular dynamics (MD) simulations are used to determine if the protein adopts a side-on, head-on, or axial orientation at the AWI with two different forcefields, GROMOS-53a6 + SPC/E and a99SB-disp + TIP4P-D. Vibrational sum frequency generation (SFG) spectroscopy experiments and spectral SFG calculations validate consistency between the structure determined from MD and experiments. Overall, we show with strong agreement that lysozyme adopts an axial conformation at pH 7. Further, we provide molecular-level insight as to how pH influences the binding domains of lysozyme resulting in side-on adsorption near the isoelectric point of the lysozyme.

Role of Surface Chemistry in the Superhydrophobicity of the Springtail Orchesella cincta (Insecta:Collembola).

Collembola are ancient arthropods living in soil with extensive exposure to dirt, bacteria, and fungi. To protect from the harsh environmental conditions and to retain a layer of air for breathing when submerged in water, they have evolved a superhydrophobic, liquid-repelling cuticle surface. The nonfouling and self-cleaning properties of springtail cuticle make it an interesting target of biomimetic materials design. Recent research has mainly focused on the intricate microstructures at the cuticle surface. Here we study the role of the cuticle chemistry for the Collembola species Orchesella cincta (Collembola, Entomobryidae). O. cincta uses a relatively simple cuticle structure with primary granules arranged to function as plastrons. In contrast to the Collembolan cuticle featuring structures on multiple length scales that is functional irrespective of surface chemistry, we found that the O. cincta cuticle loses its hydrophobic properties after being rinsed with dichloromethane. Sum frequency generation spectroscopy and time-of-flight secondary ion mass spectrometry in combination with high-resolution mass spectrometry show that a nanometer thin triacylglycerol-containing wax layer at the cuticle surface is essential for maintaining the antiwetting properties. Removal of the wax layer exposes chitin, terpenes, and lipid layers in the cuticle. With respect to biomimetic applications, the results show that, combined with a carefully chosen surface chemistry, superhydrophobicity may be achieved using a relatively unsophisticated surface structure rather than a complex, re-entrant surface structure alone.

Lasalocid Acid Antibiotic at a Membrane Surface Probed by Sum Frequency Generation Spectroscopy.

Carboxyl polyether ionophores (CPIs) are widely used as veterinary antibiotics and to increase food utilization in ruminating animals. Furthermore, CPIs can target drug-resistant bacteria, but detailed knowledge about their mode-of-action is needed to develop agents with a reasonable therapeutic index. It has been suggested that ionophores bind to membranes and incur large structural changes to shield a bound ion from the hydrophobic environment of the lipid bilayer for transport. One crucial piece of information is missing, however: Is it necessary for the free ionophore to adsorb on the membrane surface before interacting with a cation to facilitate cross-membrane ion transport? To answer this question, we applied sum-frequency generation (SFG) vibrational spectroscopy and surface tensiometry to identify the interaction between the prototypical CPI lasalocid acid (LA) and a model membrane. Observed changes in the surface pressure demonstrate that the free LA undergoes a self-assembly process with the lipid monolayer. Spectra taken from the lipid monolayer show that the free acid inserts partially into the lipid monolayer and then after complexation with sodium chloride disrupts the lipid monolayer. Overall, this study strongly suggests that this must be the crucial step of LA and metal ion complexation that allows the ionophore to traverse a lipid membrane.

Nonlinear Optical Methods for Characterization of Molecular Structure and Surface Chemistry.

The principles, strengths and limitations of several nonlinear optical (NLO) methods for characterizing biological systems are reviewed. NLO methods encompass a wide range of approaches that can be used for real-time, in-situ characterization of biological systems, typically in a label-free mode. Multiphoton excitation fluorescence (MPEF) is widely used for high-quality imaging based on electronic transitions, but lacks interface specificity. Second harmonic generation (SHG) is a parametric process that has all the virtues of the two-photon version of MPEF, yielding a signal at twice the frequency of the excitation light, which provides interface specificity. Both SHG and MPEF can provide images with high structural contrast, but they typically lack molecular or chemical specificity. Other NLO methods such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) can provide high-sensitivity imaging with chemical information since Raman active vibrations are probed. However, CARS and SRS lack interface and surface specificity. A NLO method that provides both interface/surface specificity as well as molecular specificity is vibrational sum frequency generation (SFG) spectroscopy. Vibration modes that are both Raman and IR active are probed in the SFG process, providing the molecular specificity. SFG, like SHG, is a parametric process, which provides the interface and surface specificity. SFG is typically done in the reflection mode from planar samples. This has yielded rich and detailed information about the molecular structure of biomaterial interfaces and biomolecules interacting with their surfaces. However, 2-D systems have limitations for understanding the interactions of biomolecules and interfaces in the 3-D biological environment. The recent advances made in instrumentation and analysis methods for sum frequency scattering (SFS) now present the opportunity for SFS to be used to directly study biological solutions. By detecting the scattering at angles away from the phase-matched direction even centrosymmetric structures that are isotropic (e.g., spherical nanoparticles functionalized with self-assembled monolayers or biomolecules) can be probed. Often a combination of multiple NLO methods or a combination of a NLO method with other spectroscopic methods is required to obtain a full understanding of the molecular structure and surface chemistry of biomaterials and the biomolecules that interact with them. Using the right combination methods provides a powerful approach for characterizing biological materials.

Determination of Absolute Orientation of Protein α-Helices at Interfaces Using Phase-Resolved Sum Frequency Generation Spectroscopy.

Understanding the structure of proteins at surfaces is key in fields such as biomaterials research, biosensor design, membrane biophysics, and drug design. A particularly important factor is the orientation of proteins when bound to a particular surface. The orientation of the active site of enzymes or protein sensors and the availability of binding pockets within membrane proteins are important design parameters for engineers developing new sensors, surfaces, and drugs. Recently developed methods to probe protein orientation, including immunoessays and mass spectrometry, either lack structural resolution or require harsh experimental conditions. We here report a new method to track the absolute orientation of interfacial proteins using phase-resolved sum frequency generation spectroscopy in combination with molecular dynamics simulations and theoretical spectral calculations. As a model system we have determined the orientation of a helical lysine-leucine peptide at the air-water interface. The data show that the absolute orientation of the helix can be reliably determined even for orientations almost parallel to the surface.

The Structure of the Diatom Silaffin Peptide R5 within Freestanding Two-Dimensional Biosilica Sheets.

The silaffin peptide R5 is instrumental to the mineralization of silica cell walls of diatom organisms. The peptide is also widely employed in biotechnology, for example, in the encapsulation of enzymes and for fusion proteins in tissue regeneration. Despite its scientific and technological importance, the interfacial structure of R5 during silica precipitation remains poorly understood. We herein elucidate the conformation of the peptide in its active form within silica sheets by interface-specific vibrational spectroscopy in combination with molecular dynamics simulations. Contrary to previous solution-state NMR studies, our data confirm that R5 maintains a defined structure when interacting with extended silica sheets. We show that the entire amino acid sequence of R5 interacts with silica during silica formation, leading to the intercalation of silica into the assembled peptide film.

Candle soot-based super-amphiphobic coatings resist protein adsorption.

Super nonfouling surfaces resist protein adhesion and have a broad field of possible applications in implant technology, drug delivery, blood compatible materials, biosensors, and marine coatings. A promising route toward nonfouling surfaces involves liquid repelling architectures. The authors here show that soot-templated super-amphiphobic (SAP) surfaces prepared from fluorinated candle soot structures are super nonfouling. When exposed to bovine serum albumin or blood serum, x-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry analysis showed that less than 2 ng/cm(2) of protein was adsorbed onto the SAP surfaces. Since a broad variety of substrate shapes can be coated by soot-templated SAP surfaces, those are a promising route toward biocompatible materials design.

Ice-nucleating bacteria control the order and dynamics of interfacial water.

Ice-nucleating organisms play important roles in the environment. With their ability to induce ice formation at temperatures just below the ice melting point, bacteria such as Pseudomonas syringae attack plants through frost damage using specialized ice-nucleating proteins. Besides the impact on agriculture and microbial ecology, airborne P. syringae can affect atmospheric glaciation processes, with consequences for cloud evolution, precipitation, and climate. Biogenic ice nucleation is also relevant for artificial snow production and for biomimetic materials for controlled interfacial freezing. We use interface-specific sum frequency generation (SFG) spectroscopy to show that hydrogen bonding at the water-bacteria contact imposes structural ordering on the adjacent water network. Experimental SFG data and molecular dynamics simulations demonstrate that ice-active sites within P. syringae feature unique hydrophilic-hydrophobic patterns to enhance ice nucleation. The freezing transition is further facilitated by the highly effective removal of latent heat from the nucleation site, as apparent from time-resolved SFG spectroscopy.

Kinetically Controlled Sequential Growth of Surface-Grafted Chiral Supramolecular Copolymers.

We report a facile strategy to grow supramolecular copolymers on Au surfaces by successively exposing a surface-anchored monomer to solutions of oppositely charged peptide comonomers. Charge regulation on the active chain end of the polymer sufficiently slows down the kinetics of the self-assembly process to produce kinetically trapped copolymers at near-neutral pH. We thereby achieve architectural control at three levels: The ß-sheet sequences direct the polymerization away from the surface, the height of the supramolecular copolymer brushes is well-controlled by the stepwise nature of the alternating copolymer growth, and 2D spatial resolution is realized by using micropatterned initiating monomers. The programmable nature of the resulting architectures renders this concept attractive for the development of customized biomaterials or chiral interfaces for optoelectronics and sensor applications.

IM30 triggers membrane fusion in cyanobacteria and chloroplasts.

The thylakoid membrane of chloroplasts and cyanobacteria is a unique internal membrane system harbouring the complexes of the photosynthetic electron transfer chain. Despite their apparent importance, little is known about the biogenesis and maintenance of thylakoid membranes. Although membrane fusion events are essential for the formation of thylakoid membranes, proteins involved in membrane fusion have yet to be identified in photosynthetic cells or organelles. Here we show that IM30, a conserved chloroplast and cyanobacterial protein of approximately 30 kDa binds as an oligomeric ring in a well-defined geometry specifically to membranes containing anionic lipids. Triggered by Mg(2+), membrane binding causes destabilization and eventually results in membrane fusion. We propose that IM30 establishes contacts between internal membrane sites and promotes fusion to enable regulated exchange of proteins and/or lipids in cyanobacteria and chloroplasts.

Full membrane spanning self-assembled monolayers as model systems for UHV-based studies of cell-penetrating peptides.

Biophysical studies of the interaction of peptides with model membranes provide a simple yet effective approach to understand the transport of peptides and peptide based drug carriers across the cell membrane. Herein, the authors discuss the use of self-assembled monolayers fabricated from the full membrane-spanning thiol (FMST) 3-((14-((4'-((5-methyl-1-phenyl-35-(phytanyl)oxy-6,9,12,15,18,21,24,27,30,33,37-undecaoxa-2,3-dithiahenpentacontan-51-yl)oxy)-[1,1'-biphenyl]-4-yl)oxy)tetradecyl)oxy)-2-(phytanyl)oxy glycerol for ultrahigh vacuum (UHV) based experiments. UHV-based methods such as electron spectroscopy and mass spectrometry can provide important information about how peptides bind and interact with membranes, especially with the hydrophobic core of a lipid bilayer. Near-edge x-ray absorption fine structure spectra and x-ray photoelectron spectroscopy (XPS) data showed that FMST forms UHV-stable and ordered films on gold. XPS and time of flight secondary ion mass spectrometry depth profiles indicated that a proline-rich amphipathic cell-penetrating peptide, known as sweet arrow peptide is located at the outer perimeter of the model membrane.

Reversible activation of pH-sensitive cell penetrating peptides attached to gold surfaces.

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.