Estimation of vibrational spectra of Trp-cage protein from nonequilibrium metadynamics simulations.

The development of methods that allow a structural interpretation of linear and nonlinear vibrational spectra is of great importance, both for spectroscopy and for optimizing force field quality. The experimentally measured signals are ensemble averages over all accessible configurations, which complicates spectral calculations. To account for this, we present a recipe for calculating vibrational amide-I spectra of proteins based on metadynamics molecular dynamics simulations. For each frame, a one-exciton Hamiltonian is set up for the backbone amide groups, in which the couplings are estimated with the transition-charge coupling model for nonnearest neighbors, and with a parametrized map of ab initio calculations that give the coupling as a function of the dihedral angles for nearest neighbors. The local-mode frequency variations due to environmental factors such as hydrogen bonds are modeled by exploiting the linear relationship between the amide C-O bond length and the amide-I frequency. The spectra are subsequently calculated while taking into account the equilibrium statistical weights of the frames that are determined using a previously published reweighting procedure. By implementing all these steps in an efficient Fortran code, the spectra can be averaged over very large amounts of structures, thereby extensively covering the phase space of proteins. Using this recipe, the spectral responses of 2.5 million frames of a metadynamics simulation of the miniprotein Trp-cage are averaged to reproduce the experimental temperature-dependent IR spectra very well. The spectral calculations provide new insight into the origin of the various spectral signatures (which are typically challenging to disentangle in the congested amide-I region), and allow for a direct structural interpretation of the experimental spectra and for validation of the molecular dynamics simulations of ensembles.

Probing Backbone Coupling within Hydrated Proteins with Two-Color 2D Infrared Spectroscopy.

The vibrational coupling between protein backbone modes and the role of water interactions are important topics in biomolecular spectroscopy. Our work reports the first study of the coupling between amide I and amide A modes within peptides and proteins with secondary structure and water contacts. We use two-color two-dimensional infrared (2D IR) spectroscopy and observe cross peaks between amide I and amide A modes. In experiments with peptides with different secondary structures and side chains, we observe that the spectra are sensitive to secondary structure. Water interactions affect the cross peaks, which may be useful as probes for the accessibility of protein sites to hydration water. Moving to two-color 2D IR spectra of proteins, the data demonstrate that the cross peaks integrate the sensitivities of both amide I and amide A spectra and that a two-color detection scheme may be a promising tool for probing secondary structures in proteins.

Two Receptor Binding Strategy of SARS-CoV-2 Is Mediated by Both the N-Terminal and Receptor-Binding Spike Domain.

It is not well understood why severe acute respiratory syndrome (SARS)-CoV-2 spreads much faster than other ß-coronaviruses such as SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. In a previous publication, we predicted the binding of the N-terminal domain (NTD) of SARS-CoV-2 spike to sialic acids (SAs). Here, we experimentally validate this interaction and present simulations that reveal a second possible interaction between SAs and the spike protein via a binding site located in the receptor-binding domain (RBD). The predictions from molecular-dynamics simulations and the previously-published 2D-Zernike binding-site recognition approach were validated through flow-induced dispersion analysis (FIDA)─which reveals the capability of the SARS-CoV-2 spike to bind to SA-containing (glyco)lipid vesicles, and flow-cytometry measurements─which show that spike binding is strongly decreased upon inhibition of SA expression on the membranes of angiotensin converting enzyme-2 (ACE2)-expressing HEK cells. Our analyses reveal that the SA binding of the NTD and RBD strongly enhances the infection-inducing ACE2 binding. Altogether, our work provides in silico, in vitro, and cellular evidence that the SARS-CoV-2 virus utilizes a two-receptor (SA and ACE2) strategy. This allows the SARS-CoV-2 spike to use SA moieties on the cell membrane as a binding anchor, which increases the residence time of the virus on the cell surface and aids in the binding of the main receptor, ACE2, via 2D diffusion.

Umbrella-like Helical Structure of α-Synuclein at the Air-Water Interface Observed with Experimental and Theoretical Sum Frequency Generation Spectroscopy.

The misfolding of α-synuclein (αS) into amyloid aggregates is catalyzed by hydrophobic surfaces and associated with severe brain disorders, such as Parkinson's disease. Despite the important role of interfaces, the three-dimensional structure of αS at the interfaces is still not clear. We report interface-specific sum frequency generation (SFG) experiments of monomeric αS binding to the air-water interface, a model system for the important hydrophobic surfaces. We combine the SFG spectra with calculations of theoretical spectra based on molecular dynamics simulations to show that αS, which is an intrinsically disordered protein in solution, folds into a defined, mostly helical secondary structure at the air-water interface. The binding pose resembles an umbrella shape, where the C-terminus protrudes into the water phase, while the N-terminus and the NAC region span the canopy at the interface. In this binding pose, αS is prone to aggregate, which could explain the catalytic effect of hydrophobic interfaces and air bubbles on αS fibrillation.

Liquid-Liquid Phase Separation of the Intrinsically Disordered Domain of the Fused in Sarcoma Protein Results in Substantial Slowing of Hydration Dynamics.

Formation of liquid condensates plays a critical role in biology via localization of different components or via altered hydrodynamic transport, yet the hydrogen-bonding environment within condensates, pivotal for solvation, has remained elusive. We explore the hydrogen-bond dynamics within condensates formed by the low-complexity domain of the fused in sarcoma protein. Probing the hydrogen-bond dynamics sensed by condensate proteins using two-dimensional infrared spectroscopy of the protein amide I vibrations, we find that frequency-frequency correlations of the amide I vibration decay on a picosecond time scale. Interestingly, these dynamics are markedly slower for proteins in the condensate than in a homogeneous protein solution, indicative of different hydration dynamics. All-atom molecular dynamics simulations confirm that lifetimes of hydrogen-bonds between water and the protein are longer in the condensates than in the protein in solution. Altered hydrogen-bonding dynamics may contribute to unique solvation and reaction dynamics in such condensates.

Crowding alters F-actin secondary structure and hydration.

Actin, an important component of eukaryotic cell cytoskeleton, regulates cell shape and transport. The morphology and biochemical properties of actin filaments are determined by their structure and protein-protein contacts. Crowded environments can organize filaments into bundles, but less is known about how they affect F-actin structure. This study used 2D IR spectroscopy and spectral calculations to examine how crowding and bundling impact the secondary structure and local environments in filaments and weakly or strongly bundled networks. The results reveal that bundling induces changes in actin's secondary structure, leading to a decrease in ß-sheet and an increase in loop conformations. Strongly bundled networks exhibit a decrease in backbone solvent exposure, with less perturbed α-helices and nearly "locked" ß-sheets. Similarly, the loops become less hydrated but maintain a dynamic environment. These findings highlight the role of loop structure in actin network morphology and stability under morphology control by PEG.

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.

Orientation of the Dysferlin C2A Domain is Responsive to the Composition of Lipid Membranes.

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 ß-sheets 30-40° from surface normal. However, at lipid surfaces, where PI(4,5)P2 is present, dysC2A tilts its ß-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.

In Situ Identification of Secondary Structures in Unpurified Bombyx mori Silk Fibrils Using Polarized Two-Dimensional Infrared Spectroscopy.

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 ß-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 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.

Peptide Mimic of the Marine Sponge Protein Silicatein Fabricates Ultrathin Nanosheets of Silicon Dioxide and Titanium Dioxide.

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.

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. 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.

Direct Evidence for Aligned Binding of Cellulase Enzymes to Cellulose Surfaces.

The conversion of biomass into green fuels and chemicals is of great societal interest. Engineers have been designing new cellulase enzymes for the breakdown of otherwise insoluble cellulose materials. A barrier to the rational design of new enzymes has been our lack of a molecular picture of how cellulase binding occurs. A critical factor is the attachment via the enzyme's carbohydrate binding module (CBM). To elucidate the structural and mechanistic details of cellulase adsorption, we have combined experimental data from sum frequency generation spectroscopy with molecular dynamics simulations to probe the equilibrium structure and surface alignment of a 14-residue peptide mimicking the CBM. The data show that binding is driven by hydrogen bonding and that tyrosine side chains within the CBM align the cellulase with the registry of the cellulose surface. Such an alignment is favorable for the translocation and effective cellulose breakdown and is therefore likely an important parameter for the design of novel enzymes.

Backbone Structure of Diatom Silaffin Peptide R5 in Biosilica Determined by Combining Solid-State NMR with Theoretical Sum-Frequency Generation Spectra.

Silaffin peptide R5 is key for the biogenesis of silica cell walls of diatoms. Biosilification by the R5 peptide has potential in biotechnology, drug development, and materials science due to its ability to precipitate stable, high fidelity silica sheets and particles. A true barrier for the design of novel peptide-based architectures for wider applications has been the limited understanding of the interfacial structure of R5 when precipitating silica nanoparticles. While R5-silica interactions have been studied in detail at flat surfaces, the structure within nanophase particles is still being debated. We herein elucidate the conformation of R5 in its active form within silica particles by combining interface-specific vibrational spectroscopy data with solid-state NMR torsion angles using theoretical spectra. Our calculations show that R5 is structured and undergoes a conformational transition from a strand-type motif in solution to a more curved, contracted structure when interacting with silica precursors.

In-Silico Evidence for a Two Receptor Based Strategy of SARS-CoV-2.

We propose a computational investigation on the interaction mechanisms between SARS-CoV-2 spike protein and possible human cell receptors. In particular, we make use of our newly developed numerical method able to determine efficiently and effectively the relationship of complementarity between portions of protein surfaces. This innovative and general procedure, based on the representation of the molecular isoelectronic density surface in terms of 2D Zernike polynomials, allows the rapid and quantitative assessment of the geometrical shape complementarity between interacting proteins, which was unfeasible with previous methods. Our results indicate that SARS-CoV-2 uses a dual strategy: in addition to the known interaction with angiotensin-converting enzyme 2, the viral spike protein can also interact with sialic-acid receptors of the cells in the upper airways.

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.

Direct Evidence That Mutations within Dysferlin's C2A Domain Inhibit Lipid Clustering.

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 ß-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.

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.

Unraveling VEALYL Amyloid Formation Using Advanced Vibrational Spectroscopy and Microscopy.

Intermediate species are hypothesized to play an important role in the toxicity of amyloid formation, a process associated with many diseases. This process can be monitored with conventional and two-dimensional infrared spectroscopy, vibrational circular dichroism, and optical and electron microscopy. Here, we present how combining these techniques provides insight into the aggregation of the hexapeptide VEALYL (Val-Glu-Ala-Leu-Tyr-Leu), the B-chain residue 12-17 segment of insulin that forms amyloid fibrils (intermolecularly hydrogen-bonded ß-sheets) when the pH is lowered below 4. Under such circumstances, the aggregation commences after approximately an hour and continues to develop over a period of weeks. Singular value decompositions of one-dimensional and two-dimensional infrared spectroscopy spectra indicate that intermediate species are formed during the aggregation process. Multivariate curve resolution analyses of the one and two-dimensional infrared spectroscopy data show that the intermediates are more fibrillar and deprotonated than the monomers, whereas they are less ordered than the final fibrillar structure that is slowly formed from the intermediates. A comparison between the vibrational circular dichroism spectra and the scanning transmission electron microscopy and optical microscope images shows that the formation of mature fibrils of VEALYL correlates with the appearance of spherulites that are on the order of several micrometers, which give rise to a "giant" vibrational circular dichroism effect.