Search

When a nanoparticle (NP) is introduced into a biological environment, its identity and interactions are immediately attributed to the dense layer of proteins that quickly covers the particle. The formation of this layer, dubbed the protein corona, is in general a combination of proteins interacting with the surface of the NP and a contest between other proteins for binding sites either at the surface of the NP or upon the dense layer. Despite the importance for surface engineering and drug development, the molecular mechanisms and structure behind interfacial biomolecule action have largely remained elusive. We use ultrafast sum frequency scattering (SFS) spectroscopy to determine the structure and the mode of action by which these biomolecules interact with and manipulate interfaces. The majority of work in the field of sum frequency generation has been done on flat model interfaces. This limits some important membrane properties such as membrane fluidity and dimensionality─important factors in biomolecule-membrane interactions. To move toward three-dimensional (3D) nanoscopic interfaces, we utilize SFS spectroscopy to interrogate the surface of 3D lipid monolayers, which can be used as a model lipid-based nanocarrier system. In this study, we have utilized SFS spectroscopy to follow the action of lysozyme. SFS spectra in the amide I region suggest that there is lysozyme at the interface and that the lysozyme induces an increased lipid monolayer order. The binding of lysozyme with the NP is demonstrated by an increase in acyl chain order determined by the ratio of the CH3 symmetric and CH2 symmetric peak amplitudes. Furthermore, the lipid headgroup orientation s-PO2- change strongly supports lysozyme insertion into the lipid layer causing lipid disruption and reorientation. Altogether, with SFS, we have made a huge stride toward understanding the binding and structure change of proteins within the protein corona.

Dysferlin is a 230 kD protein that plays a critical function in the active resealing of micron-sized injuries to the muscle sarcolemma by recruiting vesicles to patch the injured site via vesicle fusion. Muscular dystrophy is observed in humans when mutations disrupt this repair process or dysferlin is absent. While lipid binding by dysferlin's C2A domain (dysC2A) is considered fundamental to the membrane resealing process, the molecular mechanism of this interaction is not fully understood. By applying nonlinear surface-specific vibrational spectroscopy, we have successfully demonstrated that dysferlin's N-terminal C2A domain (dysC2A) alters its binding orientation in response to a membrane's lipid composition. These experiments reveal that dysC2A utilizes a generic electrostatic binding interaction to bind to most anionic lipid surfaces, inserting its calcium binding loops into the lipid surface while orienting its β-sheets 30-40° from surface normal. However, at lipid surfaces, where PI(4,5)P2 is present, dysC2A tilts its β-sheets more than 60° from surface normal to expose a polybasic face, while it binds to the PI(4,5)P2 surface. Both lipid binding mechanisms are shown to occur alongside dysC2A-induced lipid clustering. These different binding mechanisms suggest that dysC2A could provide a molecular cue to the larger dysferlin protein as to signal whether it is bound to the sarcolemma or another lipid surface.

We study the primary photolysis dynamics of aqueous carbonate, CO32-(aq), and hydrogen carbonate, HCO3-(aq), when they are excited at λ = 200 nm. The photolysis is recorded with sub-picosecond time resolution using UV pump-Vis probe and UV pump-IR probe transient absorption spectroscopy and interpreted with the aid of density functional theory calculations. When CO32- is excited via single photon absorption at λ = 200 nm, Φ(t = 20 ps) = 82 ± 5% of the excited di-anions either detach an electron or dissociate. The electron detachment takes place from the excited state in t < 1 ps and forms ground state CO3˙- and eaq-. Dissociation occurs from both the electronic ground and excited states of CO32-. Dissociation from the CO32- excited state is assisted by water molecules and forms CO2˙-, OH˙ and OH-. The dissociation occurs both directly from the Franck-Condon region in t < 1 ps and indirectly with a time constant of τ = 13.9 ± 0.5 ps as the excited state relaxes. Dissociation of vibrationally excited CO32- molecules in the electronic ground state is also assisted by water molecules and forms CO2 and two OH- anions. The dissociation and subsequent vibrational relaxation of CO2 occur with a time constant of τ = 10.2 ± 0.5 ps. The residual 1 - Φ(t = 20 ps) = 18 ± 5% of the excited CO32- di-anions return by internal conversion to the equilibrated CO32- ground state with a time constant of τ = 4.0 ± 0.4 ps. The extinction coefficient of aqueous hydrogen carbonate HCO3-(aq) at λ = 200 nm is an order of magnitude smaller than that of carbonate, so even though the hydrogen carbonate anions dominate the carbonate di-anions in the hydrogen carbonate solution, the primary photolysis of hydrogen carbonate is obscured by the photo-products of carbonate. Hence, we are unable to assess the primary photolysis of hydrogen carbonate. However, the weak one-photon absorption facilitates two-photon ionization of water, which forms hydronium, H3O+, cations. The sudden increase in the acidity induced by two-photon ionization protonates the ground state hydrogen carbonate molecules, thus offering a rare spectroscopic glimpse of aqueous carbonic acid.

The misfolding of ⍺-synuclein (aS) into amyloid aggregates is associated with severe brain disorders. Aggregat-ed copies of aS are found in the amyloid aggregates observed in brain tissues from Parkinson’s patients. Surfaces are known to catalyze the formation of amyloid aS aggregates. Despite the importance of the role of inter-faces and several decades of structural studies, the 3D structure of aS when bound to interfaces is still not completely clear. Hydrophobic interfaces are particularly important here. We report interface-specific sum-frequency generation (SFG) experiments to determine how monomeric aS binds to the air-water interface, a model system for hydrophobic surfaces in general. We model the SFG data by combining the experimental data directly to theoretical spectra calculations from molecular dynamics simulations. We find that aS, which is an intrinsically disordered protein in solution, folds into a defined, mostly helical, secondary structure at the air-water interface. The binding pose is reminiscent of an umbrella-shape, where the C-terminus represents the ‘pole’ and protrudes into the water phase, while the N-terminus and the NAC region span the canopy at the interface. In this binding pose, aS is prone to aggregate, which could explain the catalytic effect of hydrophobic interfaces and air bubbles on aS fibrillation.

Geckos have the astonishing ability to climb on vertical surfaces due to the adhesive properties of fibrous setae at the tips of their toe pads. While the adhesion mechanism principle, based on van der Waals interactions of myriads of spatula located at the outermost end of the setal arrays, has been studied extensively, there are still open questions about the chemistry of gecko setae. The gecko adhesive system is based on keratin fibrils assembled to support the entire setal structure. At the same time, the structure and alignment of keratin molecules within the ultrafine spatula tissue, which can support the enormous mechanical strain, still remain unknown. We have studied the molecular structure of gecko spatula using near-edge X-ray absorption fine structure (NEXAFS) imaging. We indeed found that the setae consist of a β-sheet structure aligned with the adhesion direction of the setae. Such alignment may provide mechanical stability to the setae and resistance to wear across different length scales.

We study the primary photolysis dynamics of lactic acid induced by excitation at λ = 200 nm with the aim of elucidating how simple aqueous carboxyl acids react to the deep ultraviolet exposure on the prebiotic Earth. UV-IR transient absorption spectroscopy shows a photolysis quantum yield of Φ(100 ps) = 100 ± 5%. The primary products are CO2, CO2˙− and their counter products CH3CHOH˙ and CH3CHOH−. DFT calculations suggest that the dissociation takes place from the strongly acidic nπ* excited state. Dehydroxylation of lactic acid is not observed.

The adsorption of protein to nanoparticles plays an important role in toxicity, food science, pharmaceutics, and biomaterial science. Understanding how proteins bind to nanophase surfaces is instrumental for understanding and, ultimately, controlling nanoparticle (NP) biochemistry. Techniques probing the adsorption of proteins at NP interfaces exist; however, these methods have been unable to determine the orientation and folding of proteins at these interfaces. For the first time, we probe in situ with sum frequency scattering vibrational spectroscopy the orientation of model leucine-lysine (LK) peptides adsorbed to NPs. The results show that both α-helical and β-strand LK peptides bind the particles in an upright orientation, in contrast to the flat orientation of LKs binding to planar surfaces. The different binding geometry is explained by Coulombic forces between peptides across the particle volume.

Vibrational sum frequency generation (vSFG) spectroscopy is widely used to probe the protein structure at interfaces. Because protein vSFG spectra are complex, they can only provide detailed structural information if combined with computer simulations of protein molecular dynamics and spectra calculations. We show how vSFG spectra can be accurately modeled using a surface-specific velocity-velocity scheme based on ab initio normal modes. Our calculated vSFG spectra show excellent agreement with the experimental sum frequency spectrum of LT alpha 14 peptide and provide insight into the origin of the characteristic alpha-helical amide I peak. Analysis indicates that the peak shape can be explained largely by two effects: (1) the uncoupled response of amide groups located on opposite sides of the alpha-helix will have different orientations with respect to the interface and therefore different local environments affecting the local mode vibrations and (2) vibrational splitting from nearest neighbor coupling evaluated as inter-residue vibrational correlation. The conclusion is consistent with frequency mapping techniques with an empirically based ensemble of peptide structures, thus showing how time correlation approaches and frequency mapping techniques can give independent yet complementary molecular descriptions of protein vSFG. These models reveal the sensitive relationship between protein structure and their amide I response, allowing exploitation of the complicated molecular vibrations and their interference to derive the structures of proteins under native conditions at interfaces.

Dynamin-like proteins are membrane remodeling GTPases with well-understood functions in eukaryotic cells. However, bacterial Dynamin-like proteins are still poorly investigated. SynDLP, the Dynamin-like protein of the cyanobacterium Synechocystis sp. PCC 6803, forms ordered oligomers in solution. The 3.7 Å resolution cryo-EM structure of SynDLP oligomers reveals the presence of oligomeric stalk interfaces typical for eukaryotic Dynamin-like proteins. The bundle signaling element domain shows distinct features, such as an intramolecular disulfide bridge that affects the GTPase activity, or an expanded intermolecular interface with the GTPase domain. Such atypical GTPase domain interfaces might be a GTPase activity regulating tool in oligomerized SynDLP. Furthermore, we show that SynDLP interacts with and intercalates into membranes containing negatively charged thylakoid membrane lipids. SynDLP is able to fuse membranes in a nucleotide-independent process in vitro. Thus, we assign SynDLP to the subclass of fusogenic Dynamin-like proteins.

We study the primary dissociation dynamics of aqueous formamide (HCONH2) and dimethylformamide (HCON(CH3)2) induced by photo-excitation at λ = 200 nm. The photolysis is recorded with sub-picosecond time resolution by UV pump-IR probe transient absorption spectroscopy. Formamide dissociates with a quantum yield of Φ(t = 20 ps) = 0.30 ± 0.05, t = 20 ps after the excitation. The rest of the excited formamide molecules return to the ground state within t = 1 ps and vibrationally relax towards equilibrium in t ≈ 10 ps. The only product observed is NH3. NH3 is produced with a yield of Φ(NH3) = 0.23 ± 0.10 on a timescale of τ = 3 ± 1 ps and likely constitutes the dominating product. The CO counter product to NH3 is not observed. Dimethylformamide is photolysed with a quantum yield of Φ(t = 30 ps) = 0.29 ± 0.05, t = 30 ps after the excitation. The photolysis of dimethylformamide produces CO on a time scale of τ ≈ 30 ps. The data indicate that dimethylamine and the N(CH3)2 radical are likely photoproducts.

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. Published under an exclusive license by the AVS.

Determining the secondary and tertiary structures of proteins at aqueous interfaces is crucial for understanding their function, but measuring these structures selectively at the interface is challenging. Here we demonstrate that two-dimensional infrared (2D-IR) spectroscopy of protein stabilized emulsions offers a new route to measuring interfacial protein structure with high levels of detail. We prepared hexadecane/water oil-in-water emulsions stabilized by model LK peptides that are known to fold into either alpha-helix or beta-sheet conformations at hydrophobic interfaces and measured 2D-IR spectra in a transmission geometry. We saw clear spectral signatures of the peptides folding at the interface, with no detectable residue from remaining bulk peptides. Using 2D spectroscopy gives us access to correlation and dynamics data, which enables structural assignment in cases where linear spectroscopy fails. Using the emulsions allows one to study interfacial spectra with standard transmission geometry spectrometers, bringing the richness of 2D-IR to the interface with no additional optical complexity.

Staphylococcus epidermidis causes some of the most hard-to-treat clinical infections by forming biofilms: Multicellular communities of bacteria encased in a protective matrix, supporting immune evasion and tolerance against antibiotics. Biofilms occur most commonly on medical implants, and a key event in implant colonization is the robust adherence to the surface, facilitated by interactions between bacterial surface proteins and host matrix components. S. epidermidis is equipped with a giant adhesive protein, extracellular matrix-binding protein (Embp), which facilitates bacterial interactions with surface-deposited, but not soluble fibronectin. The structural basis behind this selective binding process has remained obscure. Using a suite of single-cell and single-molecule analysis techniques, we show that S. epidermidis is capable of such distinction because Embp binds specifically to fibrillated fibronectin on surfaces, while ignoring globular fibronectin in solution. S. epidermidis adherence is critically dependent on multivalent interactions involving 50 fibronectin-binding repeats of Embp. This unusual, Velcro-like interaction proved critical for colonization of surfaces under high flow, making this newly identified attachment mechanism particularly relevant for colonization of intravascular devices, such as prosthetic heart valves or vascular grafts. Other biofilm-forming pathogens, such as Staphylococcus aureus, express homologs of Embp and likely deploy the same mechanism for surface colonization. Our results may open for a novel direction in efforts to combat devastating, biofilm-associated infections, as the development of implant materials that steer the conformation of adsorbed proteins is a much more manageable task than avoiding protein adsorption altogether.

The fascinating adhesion of gecko to virtually any material has been related to surface interactions of myriads of spatula at the tips of gecko feet. Surprisingly, the molecular details of the surface chemistry of gecko adhesion are still largely unknown. Lipids have been identified within gecko adhesive pads. However, the location of the lipids, the extent to which spatula are coated with lipids, and how the lipids are structured are still open questions. Lipids can modulate adhesion properties and surface hydrophobicity and may play an important role in adhesion. We have therefore studied the molecular structure of lipids at spatula surfaces using near-edge X-ray absorption fine structure imaging. We provide evidence that a nanometre-thin layer of lipids is present at the spatula surfaces of the tokay gecko ( Gekko gecko ) and that the lipids form ordered, densely packed layers. Such dense, thin lipid layers can effectively protect the spatula proteins from dehydration by forming a barrier against water evaporation. Lipids can also render surfaces hydrophobic and thereby support the gecko adhesive system by enhancement of hydrophobic–hydrophobic interactions with surfaces.

Two-dimensional (2D) materials have attracted attention for potential applications in light harvesting, catalysis, and molecular electronics. Mineral proteins involved in hard tissue biogenesis can produce 2D structures with high fidelity by using sustainable production routes. This study shows that a peptide mimic based on the catalytic triad of the marine sponge protein silicatein catalyzes the formation of nanometer thin and stable sheets of silicon dioxide and titanium dioxide.

The susceptibility of aqueous dipeptides to photodissociation by deep ultraviolet irradiation is studied by femtosecond spectroscopy supported by density functional theory calculations. The primary photodynamics of the aqueous dipeptides of glycyl-glycine (gly-gly), alalyl-alanine (ala-ala), and glycyl-alanine (gly-ala) show that upon photoexcitation at a wavelength of 200 nm, about 10% of the excited dipeptides dissociate by decarboxylation within 100 ps, while the rest of the dipeptides return to their native ground state. Accordingly, the vast majority of the excited dipeptides withstand the deep ultraviolet excitation. In those relatively few cases, where excitation leads to dissociation, the measurements show that deep ultraviolet irradiation breaks the Cα-C bond rather than the peptide bond. The peptide bond is thereby left intact, and the decarboxylated dipeptide moiety is open to subsequent reactions. The experiments indicate that the low photodissociation yield and in particular the resilience of the peptide bond to dissociation are due to rapid internal conversion from the excited state to the ground state, followed by efficient vibrational relaxation facilitated by intramolecular coupling among the carbonate and amide modes. Thus, the entire process of internal conversion and vibrational relaxation to thermal equilibrium on the dipeptide ground state occurs on a time scale of less than 2 ps.

We study the primary reaction dynamics of aqueous oxalate following photo-excitation of the nO→ πCO* transition atλ= 200 nm. After the excitation, some of the oxalate molecules return to the electronic ground state on two very different time scales: a fast component ofτ= 1.1 ± 0.5 ps comprising 40% of the excited molecules and a much slower component ofτ= 0.28 ± 0.05 ns accounting for 15% of the excited molecules. The remaining 45% of the excited molecules do not return to the ground state during the first 500 ps, because they either detach an electron, dissociate or stay excited for hundreds of picoseconds. Dissociation and electron detachment of oxalate predominantly produces CO2molecules with only minor yields of CO2˙−radical anions. The CO2formation is accompanied by the ejection of electrons.

We study the primary photolysis dynamics of aqueous lactate induced by photo-excitation atλ= 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 CO2and CH3CHOH−through the lowest singlet excited state of lactate, which has a lifetime ofτ= 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%.

The aberrant folding of proteins into amyloid aggregates is associated with over 50 diseases. The amyloid aggregation of α-synuclein (αS), related to Parkinson’s disease, can be catalyzed by lipid-membrane surfaces. Despite the importance of lipid surfaces and several decades of structural studies, the 3D-structure of lipid-membrane bound αS is still not known in detail. In particular, there is little information about the self-assembly and orientation of αS when interacting with lipid surfaces under physiologically relevant conditions. Here, we report interface-specific vibrational sum-frequency generation (VSFG) experiments revealing how monomeric αS binds, folds and orients at anionic lipid membranes. Since VSFG is inherently surface specific, the experiments can be performed at high αS–lipid ratios, far beyond previous structural studies. To interpret the experimental VSFG data, we present an analysis method in which out-of-equilibrium molecular-dynamics simulations are used to generate a large amount of conformations, after which their spectral match is evaluated with excitonic amide-I calculations. Hereby, the previously-determined flat-lying helical structures are corroboratively derived at low αS concentrations, while at higher, physiological concentrations, a transition to interface-protruding αS structures occurs. Such an upright conformation promotes more extensive lateral interactions between monomers and may explain how, at high protein–lipid ratios, lipid membranes can catalyze the formation of αS amyloids.

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.

The mechanical properties of biomaterials are dictated by the interactions and conformations of their building blocks, typically proteins. Although the macroscopic behaviour of biomaterials is widely studied, our understanding of the underlying molecular properties is generally limited. Among the non-invasive and label-free methods to investigate molecular structures, infrared spectroscopy is one of the most commonly used tools, because the absorption bands of the amide groups strongly depend on protein secondary structure. However, spectral congestion usually complicates the analysis of the amide spectrum. Here, we apply polarized two-dimensional (2D) infrared spectroscopy (IR) to directly identify the protein secondary structures in native silk filks cast fromBombyx morisilk feedstock. Without any additional analysis, such as peak fitting, we find that the initial effect of hydration is an increase of the random-coil content at the expense of theα-helix content, while theβ-sheet content is unchanged, and only increases at a later stage. This paper demonstrates that 2D-IR can be a valuable tool for characterizing biomaterials.

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.

Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sarcolemma resides the multidomain dysferlin protein. Mutations in this protein render it unable to repair the sarcolemma and have been linked to muscular dystrophy. A key step in dysferlin-regulated repair is the binding of the C2A domain to the lipid membrane upon increased intracellular calcium. Mutations mapped to this domain cause loss of binding ability of the C2A domain. There is a crucial need to understand the geometry of dysferlin C2A at a membrane interface as well as cell membrane lipid reorientation when compared to that of a mutant. Here, we describe a comparison between the wild-type dysferlin C2A and a mutation to the conserved aspartic acids in the domain binding loops. To identify both the geometry and the cell membrane lipid reorientation, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the interaction with a model cell membrane composed of phosphotidylserine and phosphotidylcholine. Observed changes in surface pressure demonstrate that calcium-bridged electrostatic interactions govern the initial interaction of the C2A domains docking with a lipid membrane. SFG spectra taken from the amide-I region for the wild type and variant contain features near 1642, 1663, and 1675 cm-1 related to the C2A domain β-sandwich secondary structure, indicating that the domain binds in a specific orientation. Mapping simulated SFG spectra to the experimentally collected spectra indicated that both wild-type and variant domains have nearly the same orientation to the membrane surface. However, examining the ordering of the lipids that make up a model membrane using SFG, we find that the wild type clusters the lipids as seen by the increase in the ratio of the CD3 and CD2 symmetric intensities by 170% for the wild type and by 120% for the variant. This study highlights the capabilities of SFG to probe with great detail biological mutations in proteins at cell membrane interfaces.

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.

High-resolution structural information on membrane proteins is essential for understanding cell biology and for the structure-based design of new medical drugs and drug delivery strategies. X-ray diffraction (XRD) can provide angstrom-level information about the structure of membrane proteins, yet for XRD experiments, proteins are removed from their native membrane environment, chemically stabilized, and crystallized, all of which can compromise the conformation. Here, we describe how a combination of surface-sensitive vibrational spectroscopy and molecular dynamics simulations can account for the native membrane environment. We observe the structure of a glycerol facilitator channel (GlpF), an aquaporin membrane channel finely tuned to selectively transport water and glycerol molecules across the membrane barrier. We find subtle but significant differences between the XRD structure and the inferred in situ structure of GlpF.

The mechanical properties of biomaterials are dictated by the interactions and conformations of their building blocks, typically proteins. Although the macroscopic behavior of biomaterials is widely studied, our understanding of the underlying molecular properties is generally limited. Among the noninvasive and label-free methods to investigate molecular structures, infrared spectroscopy is one of the most commonly used tools because the absorption bands of amide groups strongly depend on protein secondary structure. However, spectral congestion usually complicates the analysis of the amide spectrum. Here, we apply polarized two-dimensional (2D) infrared spectroscopy (IR) to directly identify the protein secondary structures in native silk films cast from Bombyx mori silk feedstock. Without any additional peak fitting, we find that the initial effect of hydration is an increase of the random coil content at the expense of the helical content, while the β-sheet content is unchanged and only increases at a later stage. This paper demonstrates that 2D-IR can be a valuable tool for characterizing biomaterials.

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.

Calcium oxalate (CaC2O4) is the major component of kidney stone. The acidic osteopontin (OPN) protein in human urine can effectively inhibit the growth of CaC2O4 crystals, thereby acting as a potent stone preventer. Previous studies in bulk solution all attest to the importance of binding and recognition of OPN at the CaC2O4 mineral surface, yet molecular level insights into the active interface during CaC2O4 mineralization are still lacking. Here, we probe the structure of the central OPN fragment and its interaction with Ca2+ and CaC2O4 at the water–air interface using surface‐specific non‐linear vibrational spectroscopy. While OPN peptides remain largely disordered in solution, our results reveal that the bidentate binding of Ca2+ ions refold the interfacial peptides into well‐ordered and assembled β‐turn motifs. One critical intermediate directs mineralization by releasing structural freedom of backbone and binding side chains. These insights into the mineral interface are crucial for understanding the pathological development of kidney stones and possibly relevant for calcium oxalate biomineralization in general., Surface‐specific vibrational spectroscopy was applied to study the structure and interaction of central urinary osteopontin peptide at the air–water interface and during calcium oxalate mineralization. The interfacial peptides show ordered and assembled β‐turn motifs with binding of Ca2+ ions and CaC2O4 minerals, in contrast to the disordered peptide structure in bulk solution.

Petroleum asphaltenes are surface-active compounds found in crude oils, and their interactions with surfaces and interfaces have huge implications for many facets of reservoir exploitation, including production, transportation, and oil-water separation. The asphaltene fraction in oil, found in the highest boiling-point range, is composed of many different molecules that vary in size, functionality, and polarity. Studies done on asphaltene fractions have suggested that they interact via polyaromatic and heteroaromatic ring structures and functional groups containing nitrogen, sulfur, and oxygen. However, isolating a single pure chemical structure of asphaltene in abundance is challenging and often not possible, which impairs the molecular-level study of asphaltenes of various architectures on surfaces. Thus, to further the molecular fundamental understanding, we chose to use functionalized model asphaltenes (AcChol-Th, AcChol-Ph, and 1,6-DiEtPy[Bu-Carb]) and model self-assembled monolayer (SAM) surfaces with precisely known chemical structures, whereby the hydrophobicity of the model surface is controlled. We applied solutions of asphaltenes to these SAM surfaces and then analyzed them with surface-sensitive techniques of near-edge X-ray absorption fine structure (NEXAFS) and X-ray photoelectron spectroscopy (XPS). We observe no adsorption of asphaltenes to the hydrophobic surface. On the hydrophilic surface, AcChol-Ph penetrates into the SAM with a preferential orientation parallel to the surface; AcChol-Th adsorbs in a similar manner, and 1,6-DiEtPy[Bu-Carb] binds the surface with a bent binding geometry. Overall, this study demonstrates the need for studying pure and fractionated asphaltenes at the molecular level, as even within a family of asphaltene congeners, very different surface interactions can occur.

A protein-based lubricating substance is discovered in the femoro-tibial joint of the darkling beetleZophobas morio(Insecta). The substance extrudes to the contacting areas within the joint and appears in a form of filiform flows and short cylindrical fragments. The extruded lubricating substance effectively reduces the coefficient of sliding friction to the value of 0.13 in the tribosystemglass/lubricant/glass. This value is significantly lower than 0.35 in the control tribosystemglass/glassand comparable to the value of 0.14 for the tribosystemglass/dry PTFE(polytetrafluoroethylene or Teflon). The study shows for the first time that the friction-reducing mechanism found inZ. moriofemoro-tibial joints is based on the lubricant spreading over the contacting surfaces rolling or moving at low loads and deforming at higher loads, preventing direct contact of joint counterparts. BesidesZ. morio, the lubricant has been found in the leg joints of the Argentinian wood roachBlaptica dubia.

The outermost surface of insect cuticle is a high-performance interface that provides wear protection, hydration, camouflage and sensing. The complex and inhomogeneous structure of insect cuticle imposes stringent requirements on approaches to elucidate its molecular structure and surface chemistry. Therefore, a molecular understanding and possible mimicry of the surface of insect cuticle has been a challenge. Conventional optical and electron microscopies as well as biochemical techniques provide information about morphology and chemistry but lack surface specificity. We here show that a near edge X-ray absorption fine structure microscope at the National Synchrotron Light Source can probe the surface chemistry of the curved and inhomogeneous cuticle of the African flower scarab. The analysis shows the distribution of organic and inorganic surface species while also hinting at the presence of aragonite at the dorsal protrusion region of the Eudicella gralli head, in line with its biological function., Biology serves as inspiration in materials development; this requires improved understanding of the surface chemistry responsible for processes which are being mimicked. Here, the authors report on the use of near edge X-ray absorption fine structure (NEXAFS) imaging to analyze the surface chemistry of insect cuticle.