Advantages of a synchrotron light source for fluorescence-detected linear dichroism.

Fluorescence-detected linear dichroism (FD-LD) enables one to collect linear dichroism spectra for oriented fluorophores in the presence of other absorbing species and light scattering. The experiment proceeds by scanning the excitation wavelength and using a filter to collect only emitted photons from the fluorophore. Thus, it has the potential to give data with enhanced selectivity and quality. By using a synchrotron radiation light source and fluorescence-detection, we show data for a range of fluorophores in different orienting environments. Film and flow-oriented FD-LD spectra were collected down to 170 nm. Even for flow-oriented liposomes, we have data collected down to 210 nm. For strongly scattering samples, for example, liposomes, FD-LD has the clear advantage that scattering is absent for the longer wavelength fluorescence photons. The collimated and smaller beam size of the synchrotron radiation also gives rise to sharper and more well-defined features in the spectra.

Chiroptical properties of membrane glycerophospholipids and their chiral backbones.

Glycerophospholipid membranes are one of the key cellular components. Still, their species-dependent composition and homochirality remain an elusive subject. In the context of the astrophysical circularly polarized light scenario likely involved in the generation of a chiral bias in meteoritic amino and sugar acids in space, and consequently in the origin of life's homochirality on Earth, this study reports the first measurements of circular dichroism and anisotropy spectra of a selection of glycerophospholipids, their chiral backbones and their analogs. The rather low asymmetry in the interaction of UV/VUV circularly polarized light with sn-glycerol-1/3-phosphate indicates that chiral photons would have been unlikely to directly induce symmetry breaking to membrane lipids. In contrast, the anisotropy spectra of d-3-phosphoglyceric acid and d-glyceraldehyde-3-phosphate unveil up to 20 and 100 times higher maximum anisotropy factor values, respectively. This first experimental report, targeted on investigating the origins of phospholipid symmetry breaking, opens up new avenues of research to explore alternative mechanisms leading to membrane lipid homochirality, while providing important clues for the search for chiral biosignatures of extant and/or extinct life in space, in particular for the ExoMars 2028 mission.

Uncovering the chiral bias of meteoritic isovaline through asymmetric photochemistry.

Systematic enrichments of L-amino acids in meteorites is a strong indication that biological homochirality originated beyond Earth. Although still unresolved, stellar UV circularly polarized light (CPL) is the leading hypothesis to have caused the symmetry breaking in space. This involves the differential absorption of left- and right-CPL, a phenomenon called circular dichroism, which enables chiral discrimination. Here we unveil coherent chiroptical spectra of thin films of isovaline enantiomers, the first step towards asymmetric photolysis experiments using a tunable laser set-up. As analogues to amino acids adsorbed on interstellar dust grains, CPL-helicity dependent enantiomeric excesses of up to 2% were generated in isotropic racemic films of isovaline. The low efficiency of chirality transfer from broadband CPL to isovaline could explain why its enantiomeric excess is not detected in the most pristine chondrites. Notwithstanding, small, yet consistent L-biases induced by stellar CPL would have been crucial for its amplification during aqueous alteration of meteorite parent bodies.

Surface Electronic Structure Engineering of Manganese Bismuth Tellurides Guided by Micro-Focused Angle-Resolved Photoemission.

Modification of the electronic structure of quantum matter by ad atom deposition allows for directed fundamental design of electronic and magnetic properties. This concept is utilized in the present study in order to tune the surface electronic structure of magnetic topological insulators based on MnBi2 Te4 . The topological bands of these systems are typically strongly electron-doped and hybridized with a manifold of surface states that place the salient topological states out of reach of electron transport and practical applications. In this study, micro-focused angle-resolved photoemission spectroscopy (microARPES) provides direct access to the termination-dependent dispersion of MnBi2 Te4 and MnBi4 Te7 during in situ deposition of rubidium atoms. The resulting band structure changes are found to be highly complex, encompassing coverage-dependent ambipolar doping effects, removal of surface state hybridization, and the collapse of a surface state band gap. In addition, doping-dependent band bending is found to give rise to tunable quantum well states. This wide range of observed electronic structure modifications can provide new ways to exploit the topological states and the rich surface electronic structures of manganese bismuth tellurides.

An electron-ion coincidence spectrometer for commissioning of a synchrotron radiation beamline: Absolute photon intensity and content of higher harmonic radiation.

We describe the commissioning of a new electron-ion coincidence spectrometer used to diagnose the photon beam from a plane grating monochromator beamline at the ASTRID2 synchrotron radiation facility. The spectrometer allows determination of the absolute photon intensity by calibration to the photoabsorption cross sections of known gases, such as the rare gases Ar, Kr, and Xe presented here. The spectrometer operates at very low pressure (∼10-8-10-9 mbar) and-due to the coincidence electron-ion detection scheme-the detector efficiencies can be determined routinely; hence, the spectrometer can be recalibrated swiftly. By variation of a single potential of the spectrometer, the content of higher order radiation in the monochromatized synchrotron radiation can be analyzed. The layout and operation of the synchrotron radiation beamline at ASTRID2 and its additional photon diagnostic units are additionally described.

Electronic State Spectroscopy of Nitromethane and Nitroethane.

High-resolution photoabsorption cross-sections in the 3.7-10.8 eV energy range are reinvestigated for nitromethane (CH3NO2), while for nitroethane (C2H5NO2), they are reported for the first time. New absorption features are observed for both molecules which have been assigned to vibronic excitations of valence, Rydberg, and mixed valence-Rydberg characters. In comparison with nitromethane, nitroethane shows mainly broad absorption bands with diffuse structures, which can be interpreted as a result of the side-chain effect contributing to an increased number of internal degrees of freedom. New theoretical quantum chemical calculations performed at the time-dependent density functional theory (TD-DFT) level were used to qualitatively help interpret the recorded photoabsorption spectra. From the photoabsorption cross-sections, photolysis lifetimes in the terrestrial atmosphere have been obtained for both compounds. Relevant internal conversion from Rydberg to valence character is noted for both molecules, while the nuclear dynamics of CH3NO2 and C2H5NO2 along the C-N reaction coordinate have been evaluated through potential energy curves at the TD-DFT level of theory, showing that the pre-dissociative character is more prevalent in nitromethane than in nitroethane.

Near and vacuum UV polarization spectroscopy of 1,4-distyrylbenzene.

The UV absorbance bands of 1,4-distyrylbenzene (1,4-Bis[(E)-2-phenylethenyl]benzene, DSB) are investigated by Synchrotron Radiation Linear Dichroism (SRLD) spectroscopy using stretched polyethylene as an anisotropic solvent. The observed polarization data provide information on the transition moment directions of the observed spectral features. The investigation covers the range 15,000-58,000 cm-1 (667-172 nm), thereby providing new information on the transitions of DSB in the vacuum UV region. The observed spectrum is characterized by four main band systems centered at 27,600, 41,000, 49,800, and 57,500 cm-1 (362, 244, 201, and 174 nm). In general, the observed bands and their polarization directions are well predicted by the results of quantum chemical calculations using Time-Dependent Density Functional Theory (TD-DFT) with the functional CAM-B3LYP, and with the semiempirical all-valence-electrons method LCOAO.

Chiroptical activity of gas-phase propylene oxide predicting the handedness of interstellar circular polarization in the presolar nebula.

Propylene oxide, the first chiral molecule recently detected in the interstellar medium, has once again raised the question whether biomolecular chirality might have cosmic origins. However, accurate chiroptical properties of propylene oxide in the ultraviolet spectral range necessary to suggest possible asymmetric synthetic routes in the gas phase are scarce. Here, we report on the first experimental measurements of the anisotropy spectra of gas-phase propylene oxide in the vacuum ultraviolet spectral range. Our experimental results provide novel insights into the handedness of interstellar circular polarization at the dawn of molecular evolution of our star- and planet-forming region. Besides the fundamental importance of this new investigation for understanding the origin and evolution of homochirality on Earth, our high-resolution experimental electronic circular dichroism data will inspire new efforts in quantum computational spectroscopy.

A new endstation for extreme-ultraviolet spectroscopy of free clusters and nanodroplets.

In this work, we present a new endstation for the AMOLine of the ASTRID2 synchrotron at Aarhus University, which combines a cluster and nanodroplet beam source with a velocity map imaging and time-of-flight spectrometer for coincidence imaging spectroscopy. Extreme-ultraviolet spectroscopy of free nanoparticles is a powerful tool for studying the photophysics and photochemistry of resonantly excited or ionized nanometer-sized condensed-phase systems. Here, we demonstrate this capability by performing photoelectron-photoion coincidence experiments with pure and doped superfluid helium nanodroplets. Different doping options and beam sources provide a versatile platform to generate various van der Waals clusters as well as He nanodroplets. We present a detailed characterization of the new setup and show examples of its use for measuring high-resolution yield spectra of charged particles, time-of-flight ion mass spectra, anion-cation coincidence spectra, multi-coincidence electron spectra, and angular distributions. A particular focus of the research with this new endstation is on intermolecular charge and energy-transfer processes in heterogeneous nanosystems induced by valence-shell excitation and ionization.

Structure and Protein-Protein Interactions of Ice Nucleation Proteins Drive Their Activity.

Microbially-produced ice nucleating proteins (INpro) are unique molecular structures with the highest known catalytic efficiency for ice formation. Airborne microorganisms utilize these proteins to enhance their survival by reducing their atmospheric residence times. INpro also have critical environmental effects including impacts on the atmospheric water cycle, through their role in cloud and precipitation formation, as well as frost damage on crops. INpro are ubiquitously present in the atmosphere where they are emitted from diverse terrestrial and marine environments. Even though bacterial genes encoding INpro have been discovered and sequenced decades ago, the details of how the INpro molecular structure and oligomerization foster their unique ice-nucleation activity remain elusive. Using machine-learning based software AlphaFold 2 and trRosetta, we obtained and analysed the first ab initio structural models of full length and truncated versions of bacterial INpro. The modeling revealed a novel beta-helix structure of the INpro central repeat domain responsible for ice nucleation activity. This domain consists of repeated stacks of two beta strands connected by two sharp turns. One beta-strand is decorated with a TxT amino acid sequence motif and the other strand has an SxL[T/I] motif. The core formed between the stacked beta helix-pairs is unusually polar and very distinct from previous INpro models. Using synchrotron radiation circular dichroism, we validated the ß-strand content of the central repeat domain in the model. Combining the structural model with functional studies of purified recombinant INpro, electron microscopy and modeling, we further demonstrate that the formation of dimers and higher-order oligomers is key to INpro activity. Using computational docking of the new INpro model based on rigid-body algorithms we could reproduce a previously proposed homodimer structure of the INpro CRD with an interface along a highly conserved tyrosine ladder and show that the dimer model agrees with our functional data. The parallel dimer structure creates a surface where the TxT motif of one monomer aligns with the SxL[T/I] motif of the other monomer widening the surface that interacts with water molecules and therefore enhancing the ice nucleation activity. This work presents a major advance in understanding the molecular foundation for bacterial ice-nucleation activity.

Condensation and Protection of DNA by the Myxococcus xanthus Encapsulin: A Novel Function.

Encapsulins are protein nanocages capable of harboring smaller proteins (cargo proteins) within their cavity. The function of the encapsulin systems is related to the encapsulated cargo proteins. The Myxococcus xanthus encapsulin (EncA) naturally encapsulates ferritin-like proteins EncB and EncC as cargo, resulting in a large iron storage nanocompartment, able to accommodate up to 30,000 iron atoms per shell. In the present manuscript we describe the binding and protection of circular double stranded DNA (pUC19) by EncA using electrophoretic mobility shift assays (EMSA), atomic force microscopy (AFM), and DNase protection assays. EncA binds pUC19 with an apparent dissociation constant of 0.3 ± 0.1 µM and a Hill coefficient of 1.4 ± 0.1, while EncC alone showed no interaction with DNA. Accordingly, the EncAC complex displayed a similar DNA binding capacity as the EncA protein. The data suggest that initially, EncA converts the plasmid DNA from a supercoiled to a more relaxed form with a beads-on-a-string morphology. At higher concentrations, EncA self-aggregates, condensing the DNA. This process physically protects DNA from enzymatic digestion by DNase I. The secondary structure and thermal stability of EncA and the EncA-pUC19 complex were evaluated using synchrotron radiation circular dichroism (SRCD) spectroscopy. The overall secondary structure of EncA is maintained upon interaction with pUC19 while the melting temperature of the protein (Tm) slightly increased from 76 ± 1 °C to 79 ± 1 °C. Our work reports, for the first time, the in vitro capacity of an encapsulin shell to interact and protect plasmid DNA similarly to other protein nanocages that may be relevant in vivo.

Lipid membrane-mediated assembly of the functional amyloid-forming peptide Somatostatin-14.

Membrane-mediated assembly has been well characterised for toxic amyloid species such as the amyloid-ß peptide implicated in Alzheimer's disease. However, little is known about the membrane-mediated assembly of functional-amyloid forming peptides, recently identified as a natural storage state for neuropeptide hormones in vivo. Here, we study the aggregation of somatostatin-14 (SST-14) co-incubated with model lipid membranes. Atomic force microscopy (AFM) studies confirmed that nanofibrils formed in the presence of various lipid membranes display reduced fibrillogenesis and promote the formation of non-fibrillar oligomers. Both circular dichroism (CD) and intrinsic tryptophan fluorescence studies confirmed interaction between the peptide and the lipid bilayer; this interaction appears to drive changes in membrane-mediated aggregation kinetics. We show that both the surface charge of the membrane and chain packing drive changes in the electrostatic and hydrophobic interactions between the peptide and the membrane, and hence the rate of assembly. The similarities in the effect of the lipid membrane on aggregation of functional amyloids and the more well studied toxic amyloids suggest strong aggregation modifying lipid bilayer interactions are a ubiquitous feature of all amyloid fibrils and highlight the need for further investigation as to why this leads to toxicity in some systems and not others.

The Conformation of the N-Terminal Tails of Deinococcus grandis Dps Is Modulated by the Ionic Strength.

DNA-binding proteins from starved cells (Dps) are homododecameric nanocages, with N- and C-terminal tail extensions of variable length and amino acid composition. They accumulate iron in the form of a ferrihydrite mineral core and are capable of binding to and compacting DNA, forming low- and high-order condensates. This dual activity is designed to protect DNA from oxidative stress, resulting from Fenton chemistry or radiation exposure. In most Dps proteins, the DNA-binding properties stem from the N-terminal tail extensions. We explored the structural characteristics of a Dps from Deinococcus grandis that exhibits an atypically long N-terminal tail composed of 52 residues and probed the impact of the ionic strength on protein conformation using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism and small-angle X-ray scattering. A novel high-spin ferrous iron-binding site was identified in the N-terminal tails, using Mössbauer spectroscopy. Our data reveals that the N-terminal tails are structurally dynamic and alter between compact and extended conformations, depending on the ionic strength of the buffer. This prompts the search for other physiologically relevant modulators of tail conformation and hints that the DNA-binding properties of Dps proteins may be affected by external factors.

Structure of whey protein hydrolysate used as emulsifier in wet and dried oil delivery systems: Effect of pH and drying processing.

The secondary structure of whey protein concentrate hydrolysate (WPCH), used as an emulsifier in oil delivery systems, was investigated using Synchrotron Radiation Circular Dichroism (SRCD). The effect of pH on the conformation of peptides in solution and adsorbed at the oil/water interface, as well as the thermal stability of the systems was studied. Furthermore, oil-loaded microcapsules were produced by spray-drying or electrospraying to investigate the influence of encapsulating agents (glucose syrup, maltodextrin) and drying technique on the secondary structure of WPCH at the oil/water interface. Enzymatic hydrolysis resulted in peptides with a highly unordered structure (∼60% turns and unordered regions) in solution. However, WPCH adsorption onto the oil/water interface increased the α-helical content resulting in an improved thermal stability. The encapsulating agents and spray-drying process did not modify the conformation of WPCH at the oil/water interface. Nonetheless, electrospraying affected the SRCD spectra obtained for WPCH adsorbed at the oil/water interface.

Antioxidant peptides derived from potato, seaweed, microbial and spinach proteins: Oxidative stability of 5% fish oil-in-water emulsions.

In this study, we used a combination of quantitative proteomics and bioinformatic prediction for identifying novel antioxidant peptides. Thirty-five peptides from potato, seaweed, microbial, and spinach proteins were investigated. Based on high DPPH radical scavenging activity (IC50 ≤ 16 mg/mL), metal chelation activity, isoelectric point, and high relative abundance in the parent protein sources, 11 peptides were selected. Lipid oxidation retardation was evaluated in 5% fish oil-in-water emulsions stabilized with Tween 20, where emulsion physical stability was unaffected by peptide addition. The secondary structure of selected peptides was similar in the aqueous solution and emulsions, as confirmed by synchrotron radiation circular dichroism spectroscopy. The emulsions containing the selected peptides had lower levels of hydroperoxides and volatile compounds during storage compared to the control (without peptide). This study contributes to elucidating the effect of antioxidant peptides in emulsions and demonstrates the ability of quantitative proteomics and bioinformatics prediction to identify peptides with strong antioxidant properties.

Amino acid gas phase circular dichroism and implications for the origin of biomolecular asymmetry.

Life on Earth employs chiral amino acids in stereochemical L-form, but the cause of molecular symmetry breaking remains unknown. Chiroptical properties of amino acids - expressed in circular dichroism (CD) - have been previously investigated in solid and solution phase. However, both environments distort the intrinsic charge distribution associated with CD transitions. Here we report on CD and anisotropy spectra of amino acids recorded in the gas phase, where any asymmetry is solely determined by the genuine electromagnetic transition moments. Using a pressure- and temperature-controlled gas cell coupled to a synchrotron radiation CD spectropolarimeter, we found CD active transitions and anisotropies in the 130-280 nm range, which are rationalized by ab initio calculation. As gas phase glycine was found in a cometary coma, our data may provide insights into gas phase asymmetric photochemical reactions in the life cycle of interstellar gas and dust, at the origin of the enantiomeric selection of life's L-amino acids.

Concentration and pH effect on the electronic circular dichroism and anisotropy spectra of aqueous solutions of glyceric acid calcium salt.

Electronic circular dichroism (ECD) and anisotropy spectra carry information on differential absorption of left- and right-circularly polarized light (LCPL and RCPL) by optically active compounds. This makes them powerful tools for the rapid determination of enantiomeric excesses (ee) in asymmetric synthetic and pharmaceutical chemistry, as well as for predicting the ee inducible by ultraviolet (UV) CPL. The ECD response of a chiral molecule is, however, critically dependent on the properties of the surrounding medium. Here, we report on the first ECD/anisotropy spectra of aqueous solutions of the calcium salt dihydrate of glyceric acid. A systematic study of the effect of the salt concentration and pH on the chiroptical response revealed significant changes and the appearance of a new ECD band of opposite sign. Based on the literature, this can be rationalized by the increase in the relative proportion of free glyceric acid/glycerate to Ca2+ complexes with glycerate with decreasing salt concentration or pH. Glyceric acid can be readily produced under astrophysical conditions. The anisotropy spectra of the solution containing prevalently the free form of this dihydroxy carboxylic acid resemble the ones of previously investigated aliphatic chain hydroxycarboxylic acids and proteinogenic amino acids. This indicates possible common handedness of stellar CPL-induced asymmetry in the potential comonomers of primitive proto-peptides.

Excited States of Bromopyrimidines Probed by VUV Photoabsorption Spectroscopy and Theoretical Calculations.

We report absolute photoabsorption cross sections for gas-phase 2- and 5-bromopyrimidine in the 3.7-10.8 eV energy range, in a joint theoretical and experimental study. The measurements were carried out using high-resolution vacuum ultraviolet synchrotron radiation, with quantum chemical calculations performed through the nuclear ensemble approach in combination with time-dependent density functional theory, along with additional Franck-Condon Herzberg-Teller calculations for the first absorption band (3.7-4.6 eV). The cross sections of both bromopyrimidines are very similar below 7.3 eV, deviating more substantially from each other at higher energies. In the 7.3-9.0 eV range where the maximum cross-section is found, a single and broad band is observed for 5-bromopyrimidine, while more discernible features appear in the case of 2-bromopyrimidine. Several π* ← π transitions account for the most intense bands, while weaker ones are assigned to transitions involving the nitrogen and bromine lone pairs, the antibonding σ*Br orbital, and the lower-lying Rydberg states. A detailed comparison with the available photo-absorption data of bromobenzene is also reported. We have found significant differences regarding the main absorption band, which is more peaked in bromobenzene, becoming broader and shifting to higher energies in both bromopyrimidines. In addition, there is a significant suppression of vibrational structures and of Rydberg states in the pair of isomers, most noticeably for 2-bromopyrimidine.

Graphene Oxide Layer-by-Layer Films for Sensors and Devices.

Layer-by-layer films of poly (allylamine hydrochloride) (PAH) and graphene oxide (GO) were characterized, looking at growth with the number of bilayers, morphology, and electrical properties. The PAH/GO films revealed a linear increase in absorbance with the increase in the number of deposited bilayers, allowing the determination that 10.7 ± 0.1 mg m-2 of GO is adsorbed per unit of area of each bilayer. GO absorption bands at 146, 210, 247 and 299 nm, assigned to π-π* and n-π* transitions in the aromatic ring (phenol) and of the carboxylic group, respectively, were characterized by vacuum ultraviolet spectroscopy. The morphological characterization of these films demonstrated that they are not completely uniform, with a bilayer thickness of 10.5 ± 0.7 nm. This study also revealed that the films are composed of GO and/or PAH/GO fibers and that GO is completely adsorbed on top of PAH. The electrical properties of the films reveal that PAH/GO films present a semiconductor behavior. In addition, a slight decrease in conduction was observed when films were prepared in the presence of visible light, likely due to the presence of oxygen and moisture that contributes to the damage of GO molecules.

Structural features and stability of apo- and holo-forms of a simple iron-sulfur protein.

Iron-sulfur centers are widespread in living organisms, mostly performing electron transfer functions, either in electron transfer chains or as part of multi-enzymatic complexes, while being also present in enzyme active sites, handling substrate catalysis. Rubredoxin is the simplest iron-sulfur containing protein constituted by a single polypeptide chain of 50 to 60 amino acids, of which four cysteine residues are responsible for metal binding in a tetrahedral coordination sphere. In this manuscript we explore the structure and stability of both apo- and holo-forms of a Rubredoxin from Marinobacter hydrocarbonoclasticus using Synchrotron Radiation Circular Dichroism (SRCD) in combination with other biochemical and spectroscopic techniques. The results are consistent with a holo-protein form containing a monomeric iron center with UV-visible maxima at 760, 578, 494, 386, 356 and 279 nm, an intense EPR resonance with a g value around 4.3 and Mössbauer spectroscopy parameters of δ equal to 0.69 mm/s and ΔEQ equal to 3.25 mm/s, for the ferrous reconstituted state. SRCD data, obtained for the first time for the apo-form, show a quite defined structure with ∆ε maximum at 191 nm and minima at 203 and 231 nm. Most significantly, the presence of isosbestic points at 189 and 228 nm made the interconversion between the two stable apo- and holo-form solution structures clear. SRCD temperature dependence data shows that for both forms the denaturation process proceeds through an intermediate species.