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Fibrillation process kinetic analysis of INS in H2O and D2O at 17 mg mL⁻¹ concentration. (a) The plot of ThT fluorescence spectra at the intensity of 490 nm. (b) CD spectra of INS in H2O and D2O during fibrillation. (c) The ratio of secondary structures (β-sheet, α-helix, and random coil) from analysis of CD spectrum using BeStSel server.¹ (d) TEM image after the fibrillation process (∼28 h) of INS-H2O (top) and INS-D2O (bottom)
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Amyloid proteins that undergo self-assembly to form insoluble fibrillar aggregates have attracted much attention due to their role in biological and pathological significance in amyloidosis. This study aims to understand the amyloid aggregation dynamics of insulin (INS) in H2O using two-dimensional infrared (2D-IR) spectroscopy. Conventional IR stu...
Citations
... Hence, changing the isotopic composition of the water can be used to modulate collagen-water interactions, and so study their effect on the assembly process without affecting the electrostatic interactions due to changes in the solvent dielectric constant. A significant effect of D 2 O on protein self-assembly has been recently observed for -synuclein (aS) and insulin (INS) (19,20). In these studies, it was suggested that in D 2 O specific folded structures are stabilized, accelerating (in the case of aS) or slowing down (in the case of INS) the assembly. ...
Water is known to play an important role in collagen self-assembly, but it is still largely unclear how water–collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H 2 O/D 2 O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self-assembly. We dissolve collagen in H 2 O and D 2 O and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D 2 O than in H 2 O, and collagen in D 2 O self-assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus when compared to H 2 O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D 2 O is less hydrated than in H 2 O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen–water interface, increasing the self-assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions. These results show that water acts as a mediator between collagen monomers, by modulating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self-assembly.
... 94 As with the majority of protein studies using IR spectroscopy, these real time studies of brillation employed D 2 O as a solvent to avoid the strong H-O-H bending mode of H 2 O which overlaps the amide I band. However, following the demonstration of a method that allows 2D-IR spectra of the protein amide I band to be recorded in H 2 O, 104,105 negating the need for isotopic replacement of the solvent, another study using 2D-IR to follow bril formation has shown that the kinetics are inuenced by the nature of the solvent, being slower in D 2 O as compared to H 2 O. 106 The solvent-sensitivity of the aggregation mechanism has also been detected following a study of bril formation under conditions mimicking blood serum. 107 In this case, the additional molecular components of blood serum, salts, lipids and sugars were present, but the protein component had been removed by antibody depletion and the remainder had been subject to solvent exchange into D 2 O. ...
Time resolved infrared spectroscopy of biological molecules has provided a wealth of information relating to structural dynamics, conformational changes, solvation and intermolecular interactions. Challenges still exist however arising from the wide range of timescales over which biological processes occur, stretching from picoseconds to minutes or hours. Experimental methods are often limited by vibrational lifetimes of probe groups, which are typically on the order of picoseconds, while measuring an evolving system continuously over some 18 orders of magnitude in time presents a raft of technological hurdles. In this Perspective, a series of recent advances which allow biological molecules and processes to be studied over an increasing range of timescales, while maintaining ultrafast time resolution, will be reviewed, showing that the potential for real-time observation of biomolecular function draws ever closer, while offering a new set of challenges to be overcome.
... Hence, changing the isotopic composition of the water can be used to modulate collagen-water interactions, and so study their effect on the assembly process without affecting the electrostatic interactions due to changes in the solvent dielectric constants. A significant effect of D 2 O on protein self assembly has been recently observed for αsynuclein (aS) and insulin (INS) (16,17). In these studies, it was suggested that in D 2 O specific folded structures are stabilized, accelerating (in the case of aS) or slowing down (in the case of INS) the assembly. ...
... By combining (2D-)IR with CD and MD simulations we find that water-collagen interactions are reduced in D 2 O, leading to a more stable and less water-bound structure, without altering the collagen helicity. These findings are consistent with previous studies which have shown that D 2 O is a poorer protein solvent than H 2 O, and that D 2 O favors a less water-exposed but more stable protein structure (31,(48)(49)(50)(51)(52)(53)(54)(55)(56)(57), affecting the assembly properties (16,17,58). How can a reduction in collagen hydration affect the assembly process and the fibril structure so dramatically? ...
Water is known to play an important role in collagen self assembly, but it is still largely unclear how water-collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H 2 O/D 2 O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self assembly. We dissolve collagen in H 2 O and D 2 O, and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D 2 O than in H 2 O, and collagen in D 2 O self assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus compared to H 2 O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D 2 O is less hydrated than in H 2 O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen-water interface, increasing the self assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions, which appear to be crucial in determining the acceleration of the initial nucleation rate. These results show that water acts as a mediator between collagen monomers, by moderating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self assembly.
... In the 1980s pathologists described a peripheral 'halo' surrounding Ab aggregates which was later found to be mainly water [255]. More recently such halos were found to contain soluble Ab oligomers suggesting that aggregation dynamics bear resemblance to a crystallization process and are driven primarily by energy content in the surrounding fluid medium [256][257][258][259][260]. ...
In this paper we examine 20th century conceptual developments regarding dementia and the NDs, from the first recognition of their clinical and pathological characteristics, through recognition of their molecular and cellular attributes and, ultimately, to recognition of their dynamic and vascular origins. We introduce a new energy based causal model of these disabling neurologic conditions that provides vital insights into their origins and necessary treatment. The term 'causal' implies that future treatment of these conditions necessarily entails recognition and correction of underlying energy deficits.
... [25][26][27][28][29][30][31] In particular, β -sheets are characterized by two modes at 1620-1630 cm −1 and 1680-1700 cm −1 , 29,30,32-34 and the appearance of these bands in the FTIR spectrum, and of their cross-peak pattern in the 2DIR spectrum, is a clear indication of the presence of amyloid oligomers 35 or fibrils. [11][12][13][15][16][17][18][19]36 The application of conventional and two-dimensional IR spectroscopy has led to many new insights into amyloid structure and growth. ...
... C. Application to the 2DIR spectrum of BSA amyloid/monomer mixture As was already mentioned in the introduction, two-dimensional infrared spectroscopy has become a valuable lab tool in the study of amyloid formation and structure. [11][12][13][15][16][17][18][19]36 The β -sheet diagonal and cross-peak features in the 2DIR spectrum of amyloids provide insight into their structure, [11][12][13][15][16][17][18][19] and in addition excitonic effects in the 2DIR spectrum can give insight into the size of the amyloids. 14 However, for mixed amyloid/monomer samples 2DIR spectroscopy can become difficult due to the overlap of the amyloid and the monomer 2DIR spectra. ...
... C. Application to the 2DIR spectrum of BSA amyloid/monomer mixture As was already mentioned in the introduction, two-dimensional infrared spectroscopy has become a valuable lab tool in the study of amyloid formation and structure. [11][12][13][15][16][17][18][19]36 The β -sheet diagonal and cross-peak features in the 2DIR spectrum of amyloids provide insight into their structure, [11][12][13][15][16][17][18][19] and in addition excitonic effects in the 2DIR spectrum can give insight into the size of the amyloids. 14 However, for mixed amyloid/monomer samples 2DIR spectroscopy can become difficult due to the overlap of the amyloid and the monomer 2DIR spectra. ...
Conventional and two-dimensional infrared (2D-IR) spectroscopy are well suited to study amyloid aggregates, because the amide I mode is a sensitive probe of the aggregate structure. However, these methods are not so useful to study mixtures of aggregates and monomers, which generally have overlapping amide I spectra. Here, we show that IR-Diffusion-Ordered Spectroscopy (IR-DOSY) can disentangle the contributions of protein monomers and aggregates in FTIR and 2D-IR spectra by separating the spectral contributions based on molecular size. We rely on the fact that the diffusion coefficient of a molecule is determined by its size through the Stokes-Einstein relation, and achieve sensitivity to the diffusion coefficient by creating a concentration gradient inside an infrared sample cell and tracking its equilibration in an IR-frequency resolved manner. The protein-aggregate diffusion is too slow to be experimentally observable, so instead of tracking the arrival of molecular species diffusing into the initially empty region of the sample cell, we track the depletion of the more rapidly diffusing species as they leave the sample-filled region. In this way, we can still obtain the spectrum of very slowly diffusing species, although we cannot determine their diffusion coefficient. We first demonstrate this depletion method on a mixture of two small organic molecules, and then show how it can be used to separate the spectrum of a mixture of bovine-serum-albumin amyloids and monomers into its component spectra, both in the FTIR and 2D-IR case.
... 3D-IR-DOSY is a powerful analytical tool, opening up novel applications of 2D-IR to study supramolecular complexes, self-assembly protein fibers, and amyloids. Since recent work has demonstrated the potential of 2D-IR as a biomedical and diagnostic tool, [22,[29][30][31] this new method might also contribute to further develop 2D-IR for biomedical investigations. Performing 3D-IR-DOSY experiments with IR-compatible microfluidics [32] should make the sample injection in such experiments comparatively simple. ...
Inspired by ideas from NMR, we have developed Infrared Diffusion‐Ordered Spectroscopy (IR‐DOSY), which simultaneously characterizes molecular structure and size. We rely on the fact that the diffusion coefficient of a molecule is determined by its size through the Stokes–Einstein relation, and achieve sensitivity to the diffusion coefficient by creating a concentration gradient and tracking its equilibration in an IR‐frequency resolved manner. Analogous to NMR‐DOSY, a two‐dimensional IR‐DOSY spectrum has IR frequency along one axis and diffusion coefficient (or equivalently, size) along the other, so the chemical structure and the size of a compound are characterized simultaneously. In an IR‐DOSY spectrum of a mixture, molecules with different sizes are nicely separated into distinct sets of IR peaks. Extending this idea to higher dimensions, we also perform 3D‐IR‐DOSY, in which we combine the conformation sensitivity of femtosecond multi‐dimensional IR spectroscopy with size sensitivity.
... 20 Examples include analysis of the protein content of blood serum, including quantification of the albumin to globulin ratio, measurement of low molecular weight species, and the detection of drug binding to serum proteins. [20][21][22] 2D-IR has also been used to study the kinetics of fibril formation in H 2 O, 23 complementing a number of prior studies in deuterated media, including a method employing protein-depleted serum. 7,[24][25][26] Measurements of the protein amide I band in H 2 O were based on two advantages of 2D-IR in comparison to IR absorption methods. ...
The ability of two-dimensional infrared (2D-IR) spectroscopy to measure the amide I band of proteins in H 2 O- rather than D 2 O-based solvents by evading interfering water signals has enabled in-vivo studies of proteins under physiological conditions and in biofluids . Future exploitation of 2D-IR in analytical settings, from diagnostics to protein screening, will however require comparisons between multiple datasets, necessitating control of data collection protocols to minimise measurement-to-measurement inconsistencies. Inspired by analytical spectroscopy applications in other disciplines, we describe a workflow for pre-processing 2D-IR data that aims to simplify spectral cross-comparisons. Our approach exploits the thermal water signal that is collected simultaneously with, but is temporally separated from the amide I response to guide custom baseline correction and spectral normalisation strategies before combining them with Principal Component noise reduction tools. Case studies show that application of elements of the pre-processing workflow to previously-published data enables improvements in quantification accuracy and detection limits. We subsequently apply the complete workflow in a new pilot study, testing the ability of a prototype library of 2D-IR spectra to quantify the four major protein constituents of blood serum in a single, label-free measurement. These advances show progress towards the robust data handling strategies that will be necessary for future applications of 2D-IR for pharmaceutical or biomedical applications.
... 30 2D-IR Spectroscopy is particularly well suited to study time-dependent changes in the secondary structure of proteins. 26,29,31,32 Here, we apply 2DIR spectroscopy to investigate in situ the secondary structures present in films produced using unpurified fibroin from Bombyx mori silkworms. By selecting specific polarization combinations, we obtain unique spectral signatures that allow us to disentangle and assign vibrational bands to specific secondary structures. ...
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 from Bombyx mori silk 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 the α -helix 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.
Thiocyanates, nitriles, and azides represent a versatile set of vibrational probes to measure the structure and dynamics in biological systems. The probes are minimally perturbative, the nitrile stretching mode appears in an otherwise uncongested spectral region, and the spectra report on the local environment around the probe. Nitrile frequencies and lineshapes, however, are difficult to interpret, and theoretical models that connect local environments with vibrational frequencies are often necessary. However, the development of both more accurate and intuitive models remains a challenge for the community. The present work provides an experimentally consistent collection of experimental measurements, including IR absorption and ultrafast two-dimensional infrared (2D IR) spectra, to serve as a benchmark in the development of future models. Specifically, we catalog spectra of the nitrile stretching mode of methyl thiocyanate (MeSCN) in fourteen different solvents, including non-polar, polar, and protic solvents. Absorption spectra indicate that π-interactions may be responsible for the line shape differences observed between aromatic and aliphatic alcohols. We also demonstrate that a recent Kamlet–Taft formulation describes the center frequency MeSCN. Furthermore, we report cryogenic infrared spectra that may lead to insights into the peak asymmetry in aprotic solvents. 2D IR spectra measured in protic solvents serve to connect hydrogen bonding with static inhomogeneity. We expect that these insights, along with the publicly available dataset, will be useful to continue advancing future models capable of quantitatively describing the relation between local environments, line shapes, and dynamics in nitrile probes.
