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.

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.

Structure and Dynamics of Interfacial Peptides and Proteins from Vibrational Sum-Frequency Generation Spectroscopy.

Proteins at interfaces play important roles in cell biology, immunology, bioengineering, and biomimetic material design. Many biological processes are based on interfacial protein action, ranging from cellular communication to immune responses and the protein-driven mineralization of bone. Despite the importance of interfacial proteins, comparatively little is known about their structure. The standard methods for studying crystalline or solution-phase proteins (X-ray diffraction and NMR spectroscopy) are not well-suited for studying proteins at interfaces, and for these proteins we still lack a corresponding technique that can provide the same level of structural resolution. This is not surprising in view of the challenges involved in probing the structure of proteins within monomolecular films assembled at a very thin interface in situ. Vibrational sum-frequency generation (SFG) spectroscopy has the potential to overcome this challenge and investigate the structure and dynamics of proteins at interfaces at the molecular level with subpicosecond time resolution. While SFG studies were initially limited to simple model peptides, the past decade has seen a dramatic advancement of experimental techniques and data analysis methods that has made it possible to also study interfacial proteins and their folding, binding, orientation, hydration, and dynamics. In this review, we first explain the principles of SFG spectroscopy and the experimental and theoretical methods to measure and analyze protein SFG spectra. Then we give an extensive overview of the interfacial proteins studied to date with SFG. We highlight representative examples to demonstrate recent advances in probing the structure of proteins at the interfaces of liquids, membranes, minerals, and synthetic materials.

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.

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.

Otoferlin C2F Domain-Induced Changes in Membrane Structure Observed by Sum Frequency Generation.

Proteins that contain C2 domains are involved in a variety of biological processes, including encoding of sound, cell signaling, and cell membrane repair. Of particular importance is the interface activity of the C-terminal C2F domain of otoferlin due to the pathological mutations known to significantly disrupt the protein's lipid membrane interface binding activity, resulting in hearing loss. Therefore, there is a critical need to define the geometry and positions of functionally important sites and structures at the otoferlin-lipid membrane interface. Here, we describe the first in situ probe of the protein orientation of otoferlin's C2F domain interacting with a cell membrane surface. To identify this protein's orientation at the lipid interface, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the otoferlin C2F domain interacting with model lipid membranes. A model cell membrane was built with equal amounts of phosphatidylserine and phosphatidylcholine. SFG measurements of the lipids that make up the model membrane indicate a 62% increase in amplitude from the SFG signal near 2075 cm-1 upon protein interaction, suggesting domain-induced changes in the orientation of the lipids and possible membrane curvature. This increase is related to lipid ordering caused by the docking interaction of the otoferlin C2F domain. SFG spectra taken from the amide-I region contain features near 1630 and 1670 cm-1 related to the C2F domains beta-sandwich secondary structure, thus indicating that the domain binds in a specific orientation. By mapping the simulated SFG spectra to the experimentally collected SFG spectra, we found the C2F domain of otoferlin orients 22° normal to the lipid surface. This information allows us to map what portion of the domain directly interacts with the lipid membrane.

Both Poly(ethylene glycol) and Poly(methyl ethylene phosphate) Guide Oriented Adsorption of Specific Proteins.

Developing new functional biomaterials requires the ability to simultaneously repel unwanted and guide wanted protein adsorption. Here, we systematically interrogate the factors determining the protein adsorption by comparing the behaviors of different polymeric surfaces, poly(ethylene glycol) and a poly(phosphoester), and five different natural proteins. Interestingly we observe that, at densities comparable to those used in nanocarrier functionalization, the same proteins are either adsorbed (fibrinogen, human serum albumin, and transferrin) or repelled (immunoglobulin G and lysozyme) by both polymers. However, when adsorption takes place, the specific surface dictates the amount and orientation of each protein.

Calcium-Induced Molecular Rearrangement of Peptide Folds Enables Biomineralization of Vaterite Calcium Carbonate.

Proteins can control mineralization of CaCO3 by selectively triggering the growth of calcite, aragonite or vaterite phases. The templating of CaCO3 by proteins must occur predominantly at the protein/CaCO3 interface, yet molecular-level insights into the interface during active mineralization have been lacking. Here, we investigate the role of peptide folding and structural flexibility on the mineralization of CaCO3. We study two amphiphilic peptides based on glutamic acid and leucine with ß-sheet and α-helical structures. Though both sequences lead to vaterite structures, the ß-sheets yield free-standing vaterite nanosheet with superior stability and purity. Surface-spectroscopy and molecular dynamics simulations reveal that reciprocal structuring of calcium ions and peptides lead to the effective synthesis of vaterite by mimicry of the (001) crystal plane.

Ice-binding site of surface-bound type III antifreeze protein partially decoupled from water.

Type III antifreeze proteins (AFP III) have been widely recognized as one class of ice-binding proteins produced by several biological organisms to withstand freezing conditions. Besides their ability to restrict ice growth through their ice-binding site (IBS), AFP III have also been shown to possess a great propensity for hydrophobic surfaces such as the air-water interface. Yet, it is not known whether AFP III adsorb with a specific orientation and how hydrophobic interactions affect the IBS. Molecular insights on the accessibility of the IBS and its interactions with water are important for understanding AFP III action in vivo but also for their application as ice-inhibiting agents for deicing, frozen food storage, as well as for long-term blood and organ cryo-preservation. Here, the orientation of fish AFP III adsorbed at the air-water interface has been studied using a combination of molecular dynamics (MD) simulations and vibrational sum-frequency generation (SFG) spectroscopy together with spectral calculations. The SFG/MD analysis indicated that when AFP III adsorbs at the air-water interface, it mostly retains its native state and orients with a tilt angle of 120° with respect to the surface normal. We found that the IBS is only partially solvated, leaving the pyramidal ice plane binding domain exposed to the vapor phase. These findings suggest that interactions with hydrophobic interfaces (e.g., cell membranes, polymers) could lead to the partial decoupling of the IBS from water and, to some extent, to a loss of AFP III antifreezing activity.

Hydrophobic Collapse in N-Methylacetamide-Water Mixtures.

Aqueous N-methylacetamide solutions were investigated by polarization-resolved pump-probe and 2D infrared spectroscopy (2D IR), using the amide I mode as a reporter. The 2D IR results are compared with molecular dynamics simulations and spectral calculations to gain insight into the molecular structures in the mixture. N-Methylacetamide and water molecules tend to form clusters with "frozen" amide I dynamics. This is driven by a hydrophobic collapse as the methyl groups of the N-methylacetamide molecules cluster in the presence of water. Since the studied system can be considered as a simplified model for the backbone of proteins, the present study forms a convenient basis for understanding the structural and vibrational dynamics in proteins. It is particularly interesting to find out that a hydrophobic collapse as the one driving protein folding is observed in such a simple system.

The Impact of N-terminal Acetylation of α-Synuclein on Phospholipid Membrane Binding and Fibril Structure.

Human α-synuclein (αS) has been shown to be N terminally acetylated in its physiological state. This modification is proposed to modulate the function and aggregation of αS into amyloid fibrils. Using bacterially expressed acetylated-αS (NTAc-αS) and endogenous αS (Endo-αS) from human erythrocytes, we show that N-terminal acetylation has little impact on αS binding to anionic membranes and thus likely not relevant for regulating membrane affinity. N-terminal acetylation does have an effect on αS aggregation, resulting in a narrower distribution of the aggregation lag times and rates. 2D-IR spectra show that acetylation changes the secondary structure of αS in fibrils. This difference may arise from the slightly higher helical propensity of acetylated-αS in solution leading to a more homogenous fibril population with different fibril structure than non-acetylated αS. We speculate that N-terminal acetylation imposes conformational restraints on N-terminal residues in αS, thus predisposing αS toward specific interactions with other binding partners or alternatively decrease nonspecific interactions.

C-Terminal Truncated α-Synuclein Fibrils Contain Strongly Twisted ß-Sheets.

C-terminal truncations of monomeric wild-type alpha-synuclein (henceforth WT-αS) have been shown to enhance the formation of amyloid aggregates both in vivo and in vitro and have been associated with accelerated progression of Parkinson's disease (PD). The correlation with PD may not solely be a result of faster aggregation, but also of which fibril polymorphs are preferentially formed when the C-terminal residues are deleted. Considering that different polymorphs are known to result in distinct pathologies, it is important to understand how these truncations affect the organization of αS into fibrils. Here we present high-resolution microscopy and advanced vibrational spectroscopy studies that indicate that the C-terminal truncation variant of αS, lacking residues 109-140 (henceforth referred to as 1-108-αS), forms amyloid fibrils with a distinct structure and morphology. The 1-108-αS fibrils have a unique negative circular dichroism band at ∼230 nm, a feature that differs from the canonical ∼218 nm band usually observed for amyloid fibrils. We show evidence that 1-108-αS fibrils consist of strongly twisted ß-sheets with an increased inter-ß-sheet distance and a higher solvent exposure than WT-αS fibrils, which is also indicated by the pronounced differences in the 1D-IR (FTIR), 2D-IR, and vibrational circular dichroism spectra. As a result of their distinct ß-sheet structure, 1-108-αS fibrils resist incorporation of WT-αS monomers.

Repelling and ordering: the influence of poly(ethylene glycol) on protein adsorption.

Development of new materials for drug delivery and biosensing requires the fine-tuning of interfacial properties. We report here the influence of the poly(ethylene glycol) (PEG) grafting density in model phospholipid monolayers on the adsorption behavior of bovine serum albumin and human fibrinogen, not only with respect to the amount of adsorbed protein, but also its orientational ordering on the surface. As expected, with increasing interfacial PEG density, the amount of adsorbed protein decreases up to the point where complete protein repellency is reached. However, at intermediate concentrations, the net orientation of adsorbed fibrinogen is highest. The different proteins respond differently to PEG, not only in the amount of protein adsorbed, but also in the manner that proteins adsorb. The results show that for specific cases, tuning the interfacial PEG concentration allows to guide the protein adsorption configuration, a feature sought after in materials for both biosensing and biomedical applications.

Sticky water surfaces: helix-coil transitions suppressed in a cell-penetrating peptide at the air-water interface.

GALA is a 30 amino acid synthetic peptide consisting of a Glu-Ala-Leu-Ala repeat and is known to undergo a reversible structural transition from a disordered to an α-helical structure when changing the pH from basic to acidic values. In its helical state GALA can insert into and disintegrate lipid membranes. This effect has generated much interest in GALA as a candidate for pH triggered, targeted drug delivery. GALA also serves as a well-defined model system to understand cell penetration mechanisms and protein folding triggered by external stimuli. Structural transitions of GALA in solution have been studied extensively. However, cell penetration is an interfacial effect and potential biomedical applications of GALA would involve a variety of surfaces, e.g., nanoparticles, lipid membranes, tubing, and liquid-gas interfaces. Despite the apparent importance of interfaces in the functioning of GALA, the effect of surfaces on the reversible folding of GALA has not yet been studied. Here, we use sum frequency generation vibrational spectroscopy (SFG) to probe the structural response of GALA at the air-water interface and IR spectroscopy to follow GALA folding in bulk solution. We combine the SFG data with molecular dynamics simulations to obtain a molecular-level picture of the interaction of GALA with the air-water interface. Surprisingly, while the fully reversible structural transition was observed in solution, at the water-air interface, a large fraction of the GALA population remained helical at high pH. This "stickiness" of the air-water interface can be explained by the stabilizing interactions of hydrophobic leucine and alanine side chains with the water surface.

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.

Elucidating the backbone conformation of photoswitchable foldamers using vibrational circular dichroism.

The backbone conformation of amphiphilic oligo(azobenzene) foldamers is investigated using vibrational circular dichroism (VCD) spectroscopy on a mode involving the stretching of the N=N bonds in the backbone. From denaturation experiments, we find that the VCD response in the helical conformation arises mainly from through-space interaction between the N=N-stretch transition-dipole moments, so that the coupled-oscillator model can be used to predict the VCD spectrum associated with a particular conformation. Using this approach, we elucidate the origin of the VCD signals in the folded conformation, and can assign the observed partial loss of VCD signals upon photo-induced unfolding to specific conformational changes. Our results show that the N=N-stretch VCD response provides an excellent probe of the helical conformation of the N=N bonds in this type of switchable molecular system.

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.

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.

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.