Elevated concentrations cause upright alpha-synuclein conformation at lipid interfaces.

The amyloid aggregation of α-synuclein (αS), related to Parkinson's disease, can be catalyzed by lipid membranes. Despite the importance of lipid surfaces, the 3D-structure and orientation of lipid-bound αS is still not known in detail. Here, we report interface-specific vibrational sum-frequency generation (VSFG) experiments that reveal how monomeric αS binds to an anionic lipid interface over a large range of αS-lipid ratios. To interpret the experimental data, we present a frame-selection method ("ViscaSelect") in which out-of-equilibrium molecular dynamics simulations are used to generate structural hypotheses that are compared to experimental amide-I spectra via excitonic spectral calculations. At low and physiological αS concentrations, we derive flat-lying helical structures as previously reported. However, at elevated and potentially disease-related concentrations, a transition to interface-protruding αS structures occurs. Such an upright conformation promotes lateral interactions between αS monomers and may explain how lipid membranes catalyze the formation of αS amyloids at elevated protein concentrations.

Tutorials in vibrational sum frequency generation spectroscopy. III. Collecting, processing, and analyzing vibrational sum frequency generation spectra.

In this Tutorial series, we aim to provide an accessible introduction to vibrational sum frequency generation (VSFG) spectroscopy, targeted toward people entering the VSFG world without a rigorous formal background in optical physics or nonlinear spectroscopy. In this article, we discuss in detail the processes of collecting and processing VSFG data, and user-friendly processing software (sfgtools) is provided for use by people new to the field. Some discussion of analyzing VSFG spectra is also given, specifically with a discussion of fitting homodyne VSFG spectra, and a discussion of what can be learned (both qualitatively and quantitatively) from VSFG spectra.

X3Σg- → b1Σg+ Absorption Spectra of Molecular Oxygen in Liquid Organic Solvents at Atmospheric Pressure.

Spectra and absorption coefficients of the forbidden 765 nm X3Σg- → b1Σg+ transition of molecular oxygen dissolved in organic solvents at atmospheric pressure were recorded over a 5 m path length using a liquid waveguide capillary cell. The results show that it is possible to investigate this weak near-infrared absorption transition in a common liquid hydrocarbon solvent without the need for a potentially dangerous high oxygen pressure. Proof-of-principle data from benzene, toluene, chlorobenzene, bromobenzene, and iodobenzene reveal a pronounced heavy atom effect on this spin-forbidden transition. For example, the absorption coefficient at the band maximum in iodobenzene, (28.9 ± 3.3) × 10-3 M-1 cm-1, is approximately 21 times larger than that in benzene, (1.4 ± 0.1) × 10-3 M-1 cm-1. These absorption measurements corroborate results obtained from O2(X3Σg-) → O2(b1Σg+) excitation spectra of O2(a1Δg) → O2(X3Σg-) phosphorescence, which depended on data from a plethora of convoluted experiments. Spectroscopic studies of molecular oxygen in liquid solvents can help evaluate aspects of the seminal Strickler-Berg approach to treat the effect of solvent on Einstein's A and B coefficients for radiative transitions. In particular, our present results are a key step toward using the O2(X3Σg-) → O2(b1Σg+) transition to evaluate the speculated limiting condition of applying the Strickler-Berg treatment to a highly forbidden process. This latter issue is but one example of how an arguably simple homonuclear diatomic molecule continues to aid the scientific community by providing fundamental physical insight.

The Diatom Peptide R5 Fabricates Two-Dimensional Titanium Dioxide Nanosheets.

Diatoms use peptides based on the protein silaffin to fabricate their silica cell walls. To the interest of material scientists, silaffin peptides can also produce titanium dioxide nanoparticles. Peptide-based synthesis could present an environmentally friendly route to the synthesis of titanium dioxide nanomaterials with potential applications in water splitting and for biocompatible materials design. Two-dimensional nanomaterials have exceptional surface-to-volume ratios and are particularly suited for these applications. We here demonstrate how the silaffin peptide R5 can precipitate free-standing and self-supported sheets of titanium dioxide at the air-water interface, which are stable over tens of micrometers.

Structure and Orientation of the SARS-Coronavirus-2 Spike Protein at Air-Water Interfaces.

The SARS coronavirus 2 (SARS-CoV-2) spike protein is located at the outermost perimeter of the viral envelope and is the first component of the virus to make contact with surrounding interfaces. The stability of the spike protein when in contact with surfaces plays a deciding role for infection pathways and for the viability of the virus after surface contact. While cryo-EM structures of the spike protein have been solved with high resolution and structural studies in solution have provided information about the secondary and tertiary structures, only little is known about the folding when adsorbed to surfaces. We here report on the secondary structure and orientation of the S1 segment of the spike protein, which is often used as a model protein for in vitro studies of SARS-CoV-2, at the air-water interface using surface-sensitive vibrational sum-frequency generation (SFG) spectroscopy. The air-water interface plays an important role for SARS-CoV-2 when suspended in aerosol droplets, and it serves as a model system for hydrophobic surfaces in general. The SFG experiments show that the S1 segment of the spike protein remains folded at the air-water interface and predominantly binds in its monomeric state, while the combination of small-angle X-ray scattering and two-dimensional infrared spectroscopy measurements indicate that it forms hexamers with the same secondary structure in aqueous solution.

Tutorials in vibrational sum frequency generation spectroscopy. I. The foundations.

Interfaces between bulk media are often where critical molecular processes occur that can dictate the chemistry of an entire macroscopic system. Optical spectroscopy such as IR or Raman spectroscopy is often challenging to apply to interfaces due to contributions from bulk phases that dominate the spectra, masking any detail about the interfacial layer. Vibrational sum frequency generation (VSFG) spectroscopy is a nonlinear spectroscopy that allows vibrational spectra of molecules at interfaces to be directly measured. This Tutorial series is aimed at people entering the VSFG world without a rigorous formal background in optical physics or nonlinear spectroscopy. In this article, we present the fundamental theory of VSFG spectroscopy, with a focus on qualitative, intuitive explanation of the relevant physical phenomena, with minimal mathematics, to enable a newcomer to VSFG spectroscopy to quickly become conversant in the language and fundamental physics of the technique.

Tutorials in vibrational sum frequency generation spectroscopy. II. Designing a broadband vibrational sum frequency generation spectrometer.

In this Tutorial series, we aim to provide an accessible introduction to vibrational sum frequency generation (VSFG) spectroscopy, targeted toward people entering the VSFG world without a rigorous formal background in optical physics or nonlinear spectroscopy. In this article, we describe in depth how a broadband VSFG spectrometer is designed and constructed, using the instrument in SurfLab, Aarhus University, as an illustrative case. Detailed information about specific instrumentation (together with reasons why things are the way they are) is given throughout. This information is often omitted in other descriptions of such instrumentation and so will be invaluable to people new to the field.

Electrostatics Trigger Interfacial Self-Assembly of Bacterial Ice Nucleators.

Ice active bacteria can catalyze water freezing at high subzero temperatures using ice nucleating proteins (INPs) located at their outer cell walls. INPs are the most effective ice nucleators known and are of significant interest for agriculture, climate research, and freeze/antifreeze technologies. The aggregation of INPs into large ice nucleation sites is a key step for effective ice nucleation. It has been proposed that ice active bacteria can drive the aggregation of INPs and thereby trigger ice nucleation. However, the mechanism of INP aggregate assembly and the molecular processes behind the activation are still unclear. Both biochemical pathways and activation through electrostatics have been proposed based on experiments with lysed ice active bacteria. For a more direct view on the assembly of INPs, we follow the structure and water interactions of a synthetic model INP of the well-studied ice bacterium Pseudomonas syringae at the air-water interface as a function of the subphase pH. By combining sum frequency generation spectroscopy with two-dimensional infrared spectra, we conclude that self-assembly and electrostatic interactions drive the formation of ordered INP structures capable of aligning interfacial water.

A liquid surface height controller for surface spectroscopy.

We present a simple and inexpensive liquid surface height controller that can monitor and maintain the height of a liquid surface in a surface-sensitive experiment. The system is based on a commercial laser pointer, universal serial bus webcam, syringe pump, and homemade control software. The system can sense changes in the height of the surface of ±1 µm, and the maximum range of the device without readjustment is around 2.5 mm. The intended use of the device is to maintain the height of a sample at the air-water interface in a sum-frequency generation spectroscopy measurement, which constantly changes due to water evaporation. A demonstration of the system maintaining the height of a water surface to a tolerance of ±5 µm over a period of 8 h is shown to illustrate the stability of a system controlled by this device.

Photophysics of a protein-bound derivative of malachite green that sensitizes the production of singlet oxygen.

Genetically encodable proteins that photosensitize the production of singlet oxygen, O2(a1Δg), will play an increasingly important role in elucidating mechanisms of cellular processes modulated by reactive oxygen species, ROS, and changes in redox balance. In the development of such tools, it is essential to characterize the oxygen-dependent photophysics of the protein-encased chromophore. Of the O2(a1Δg)-photosensitizing systems recently developed, a protein-bound derivative of Malachite Green has several desirable features: (1) it absorbs light at wavelengths longer than those typically absorbed by endogenous molecules, and (2) the chromophore becomes a viable sensitizer only when bound to the activating protein. However, we now demonstrate that the photophysics of this Malachite Green system is not simple. Our data indicate that, with an increase in the concentration of ground-state oxygen, O2(X3Σg-), the yield of O2(a1Δg) does not increase in a proportional manner. Moreover, the lifetime of O2(a1Δg) decreases as the O2(X3Σg-) concentration is increased. One mechanism that could account for our observations involves the concomitant photo-initiated formation of O2(a1Δg) and the superoxide radical anion. We propose that the superoxide ion acts as a dynamic diffusion-dependent quencher to influence the O2(a1Δg) lifetime and as a static quencher within the protein enclosure to influence the measured O2(a1Δg) yield. Thus, in the least, caution should be exercised when using this Malachite Green system to probe mechanisms of ROS-mediated processes. Our results contribute to a better understanding of the general photophysics of protein-bound O2(a1Δg) sensitizers which, in turn, facilitates the further development of these useful mechanistic tools.

The primary photo-dissociation dynamics of lactate in aqueous solution: decarboxylation prevents dehydroxylation.

We study the primary photolysis dynamics of aqueous lactate induced by photo-excitation at λ = 200 nm. Our calculations indicate that both decarboxylation and dehydroxylation are energetically possible, but decarboxylation is favoured dynamically. UV pump - IR probe transient absorption spectroscopy shows that the photolysis is dominated by decarboxylation, whereas dehydroxylation is not observed. Analysis of the transient IR spectrum suggests that photo-dissociation of lactate primarily produces CO2 and CH3CHOH- through the lowest singlet excited state of lactate, which has a lifetime of τ = 11 ps. UV pump - VIS probe transient absorption spectroscopy of electrons from the dissociating lactate anion indicates that the anionic electron from the CO2˙- fragment is transferred to the CH3CHOH˙ counter radical during the decarboxylation process, and CO2˙- is consequently only observed as a minor photo-product. The photo-dissociation quantum yield after the full decay of the excited state is Φ(100ps) = 38 ± 5%.

Ice-nucleating proteins are activated by low temperatures to control the structure of interfacial water.

Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Using specialized ice-nucleating proteins (INPs), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds, which may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INP-induced freezing have remained largely elusive. We investigate the structural basis for the interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae. Using vibrational sum-frequency generation (SFG) and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a ß-helical structure in solution and at water surfaces. In this configuration, interaction between INPs and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with molecular-dynamics simulations and spectral calculations show that InaZ reorients at lower temperatures. This reorientation can enhance water interactions, and thereby the effectiveness of ice nucleation.

Assembly of iron oxide nanosheets at the air-water interface by leucine-histidine peptides.

The fabrication of inorganic nanomaterials is important for a wide range of disciplines. While many purely inorganic synthetic routes have enabled a manifold of nanostructures under well-controlled conditions, organisms have the ability to synthesize structures under ambient conditions. For example, magnetotactic bacteria, can synthesize tiny 'compass needles' of magnetite (Fe3O4). Here, we demonstrate the bio-inspired synthesis of extended, self-supporting, nanometer-thin sheets of iron oxide at the water-air interface through self-assembly using small histidine-rich peptides.

Synergistic effect of carotenoid and silicone-based additives for photooxidatively stable organic solar cells with enhanced elasticity.

Photochemical and mechanical stability are critical in the production and application of organic solar cells. While these factors can individually be improved using different additives, there is no example of studies on the combined effects of such additive-assisted stabilization. In this study, the properties of PTB7:[70]PCBM organic solar cells are studied upon implementation of two additives: the carotenoid astaxanthin (AX) for photochemical stability and the silicone polydimethylsiloxane (PDMS) for improved mechanical properties. A newly designed additive, AXcPDMS, based on astaxanthin covalently bonded to PDMS was also examined. Lifetime tests, produced in ISOS-L-2 conditions, reveal an improvement in the accumulated power generation (APG) of 10% with pure AX, of 90% when AX is paired with PDMS, and of 140% when AXcPDMS is added in the active layer blend, as compared to the control devices. Singlet oxygen phosphorescence measurements are utilized to study the ability of AX and AXcPDMS to quench singlet oxygen and its precursors in the films. The data are consistent with the strong stabilization effect of the carotenoids. While AX and AXcPDMS are both efficient photochemical stabilizers, the improvement in device stability observed in the presence of AXcPDMS is likely due to a more favorable localization of the stabilizer within the blend. The mechanical properties of the active layers were investigated by tensile testing and cohesive fracture measurements, showing a joint improvement of the photooxidative stability and the mechanical properties, thus yielding organic solar cell devices that are promising for flexible photovoltaic applications.

Oxygen- and pH-Dependent Photophysics of Fluorinated Fluorescein Derivatives: Non-Symmetrical vs. Symmetrical Fluorination.

Fluorescein, and derivatives of fluorescein, are often used as fluorescent probes and sensors. In systems where pH is a variable, protonation/deprotonation of the molecule can influence the pertinent photophysics. Fluorination of the xanthene moiety can alter the molecule's pKa such as to render a probe whose photophysics remains invariant over a wide pH range. Di-fluorination is often sufficient to accomplish this goal, as has been demonstrated with compounds such as Oregon Green in which the xanthene moiety is symmetrically difluorinated. In this work, we synthesized a non-symmetrical difluorinated analog of Oregon Green which we call Athens Green. We ascertained that the photophysics and photochemistry of Athens Green, including the oxygen-dependent photophysics that results in the sensitized production of singlet oxygen, O2(a1Δg), can differ appreciably from the photophysics of Oregon Green. Our data indicate that Athens Green will be a more benign fluorescent probe in systems that involve the production and removal of O2(a1Δg). These results expand the available options in the toolbox of fluorescein-based fluorophores.

Oxygen-dependent photophysics and photochemistry of prototypical compounds for organic photovoltaics: inhibiting degradation initiated by singlet oxygen at a molecular level.

Photo-initiated, oxygen-mediated degradation of the molecules in the active layer of organic photovoltaic, OPV, devices currently limits advances in the development of solar cells. To address this problem systematically and at a molecular level, it is informative to quantify the kinetics of the pertinent processes, both in solution phase and in solid films. To this end, we examined the oxygen-dependent photophysics and photochemistry of selected functionalized fullerenes, thiophene derivatives, and a subphthalocyanine commonly used in OPV devices. We find that the photosensitized production of singlet molecular oxygen, O2(a1Δg), by these molecules is a key step in the degradation process. We demonstrate that the addition of either ß-carotene or astaxanthin as antioxidants can inhibit degradation by a combination of three processes: (a) deactivation of O2(a1Δg) to the oxygen ground state, O2(X3Σg -), (b) quenching of the O2(a1Δg) precursor, and (c) sacrificial reactions of the carotenoid with free radicals formed in the photo-initiated reactions. For OPV systems in which reaction with O2(a1Δg) contributes to the degradation, the first two of these processes are desired and should have appreciable impact in prolonging the longevity of OPV devices because they do not result in a chemical change of the system.

Biomimetic Approach to Inhibition of Photooxidation in Organic Solar Cells Using Beta-Carotene as an Additive.

Recent efficiency records of organic photovoltaics (OPV) highlight stability as a limiting weakness. Incorporation of stabilizers is a desirable approach for inhibiting degradation-it is inexpensive and readily up-scalable. However, to date, such additives have had limited success. We show that ß-carotene (BC), an inexpensive and green, naturally occurring antioxidant, dramatically improves OPV stability. When compared to nonstabilized reference devices, the accumulated power generation of PTB7:[70]PCBM devices in the presence of BC increases by an impressive factor of 6, due to stabilization of both the burn-in and the lifetime, and by a factor of 21 for P3HT:[60]PCBM devices, owing to a longer lifetime. Using electron spin resonance and time-resolved near-IR emission spectroscopies, we probed radical and singlet oxygen concentrations. We demonstrate that singlet oxygen sensitized by [70]PCBM causes the "burn-in" of PTB7:[70]PCBM devices and that BC effectively mitigates it. Our results provide an effective solution to the problem that currently limits widespread use of OPV.

Single mutation in a novel bacterial LOV protein yields a singlet oxygen generator.

Mr4511 from Methylobacterium radiotolerans is a 164 amino acid protein built of a flavin mononucleotide (FMN) binding, blue-light responsive LOV (Light, Oxygen, Voltage) core domain plus flanking regions. In contrast to the majority of LOV domains, Mr4511 lacks a tryptophan residue that was previously identified as a major quencher for the FMN triplet state in photosensitizers for singlet oxygen (SO) engineered from these photoreceptors. Here we show that for Mr4511 it is sufficient to only mutate the reactive cysteine responsible for the photocycle (Cys71) in the native protein to generate an efficient SO photosensitizer: both C71S and C71G variants exhibit SO quantum yields of formation, ΦΔ, around 0.2 in air-saturated solutions. Under oxygen saturated conditions, ΦΔ reaches ∼0.5 in deuterated buffer. The introduction of Trp112 in the canonical position for LOV domains dramatically lowers ΦΔ to values comparable to miniSOG, one of the early FMN binding proteins touted as a SO sensitizer. Besides its SO properties, Mr4511 is also exceedingly robust against denaturation with urea and is more photostable than free FMN.