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&rsquo;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. &nbsp;</p

Evidence for Intramolecular Antiparallel Beta-Sheet Structure in Alpha-Synuclein Fibrils from a Combination of Two-Dimensional Infrared Spectroscopy and Atomic Force Microscopy

The aggregation of the intrinsically disordered protein alpha-synuclein (αS) into amyloid fibrils is thought to play a central role in the pathology of Parkinson's disease. Using a combination of techniques (AFM, UV-CD, XRD, and amide-I 1D- and 2D-IR spectroscopy) we show that the structure of αS fibrils varies as a function of ionic strength: fibrils aggregated in low ionic-strength buffers ([NaCl] ≤ 25 mM) have a significantly different structure than fibrils grown in higher ionic-strength buffers. The observations for fibrils aggregated in low-salt buffers are consistent with an extended conformation of αS molecules, forming hydrogen-bonded intermolecular β-sheets that are loosely packed in a parallel fashion. For fibrils aggregated in high-salt buffers (including those prepared in buffers with a physiological salt concentration) the measurements are consistent with αS molecules in a more tightly-packed, antiparallel intramolecular conformation, and suggest a structure characterized by two twisting stacks of approximately five hydrogen-bonded intermolecular β-sheets each. We find evidence that the high-frequency peak in the amide-I spectrum of αS fibrils involves a normal mode that differs fundamentally from the canonical high-frequency antiparallel β-sheet mode. The high sensitivity of the fibril structure to the ionic strength might form the basis of differences in αS-related pathologies

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

C-Terminal Truncated α-Synuclein Fibrils Contain Strongly Twisted β-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 β-sheets with an increased inter-β-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 β-sheet structure, 1-108-αS fibrils resist incorporation of WT-αS monomers

Observation of pH-Induced Protein Reorientation at the Water Surface

Hydrophobins are surface-active proteins that form a hydrophobic, water-repelling film around aerial fungal structures. They have a compact, particle-like structure, in which hydrophilic and hydrophobic regions are spatially separated. This surface property renders them amphiphilic and is reminiscent of synthetic Janus particles. Here we report surface-specific chiral and nonchiral vibrational sum-frequency generation spectroscopy (VSFG) measurements of hydrophobins adsorbed to their natural place of action, the air–water interface. We observe that hydrophobin molecules undergo a reversible change in orientation (tilt) at the interface when the pH is varied. We explain this local orientation toggle from the modification of the interprotein interactions and the interaction of hydrophobin with the water solvent, following the pH-induced change of the charge state of particular amino acids

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 β-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

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 β-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. </p