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Supramolecular chirality of amyloid fibrils, protein aggregates related to many neurodegenerative diseases, is a remarkable property associated with fibril structure and polymorphism. Since its discovery almost 10 years ago there is still little understanding of this phenomenon, including the cause of the highly enhanced vibrational circular dichroism (VCD) intensity arising from fibril supramolecular chirality. In this study, VCD spectra, enhanced by filament supramolecular chirality, are presented for lysozyme and insulin fibrils above and below pH 2 and after deuterium exchange, above and below pD 2. Supramolecular chirality (observed by VCD) and fibril morphology (documented by atomic force microscopy) are not affected by protein deuteriation. In D2 O the fibril VCD sign pattern changes to fewer bands, with implications for the amide I/II origin of enhanced VCD intensity. Separation of amide I and II signals will facilitate calculations of enhanced VCD spectra of amyloid fibrils and enable a better understanding of the origin of the VCD sign pattern.
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Amiloide/química , Dicroísmo Circular , Deutério/química , Agregados Proteicos , Animais , Humanos , Concentração de Íons de Hidrogênio , Insulina/química , Muramidase/químicaRESUMO
The mechanical properties of agarose-derived hydrogels depend on the scaffolding of the polysaccharide network. To identify and quantify such higher order structure, we applied Raman optical activity (ROA)-a spectroscopic technique that is highly sensitive toward carbohydrates-on native agarose and chemically modified agarose in the gel phase for the first time. By spectral global fitting, we isolated features that change as a function of backbone carboxylation (28, 40, 50, 60, 80, and 93 %) from other features that remain unchanged. We assigned these spectral features by comparison to ROA spectra calculated for different oligomer models. We found a 60:40 ratio of double- and single-stranded α-helix in the highly rigid hydrogel of native agarose, while the considerably softer hydrogels made from carboxylated agarose use a scaffold of unpaired ß-strands.
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Searching the 3D structural fragments of organic molecules is challenging because of structural differences between X-ray and theoretically calculated geometries and the conformational flexibility of substituents. The codification program called Conformational Code for Organic Molecules (CCOM) can be used to unambiguously convert 3D conformational data for various molecules to 1D data. Two deviations from Rule E-5.6 of the International Union of Pure and Applied Chemistry (IUPAC) Rules for Nomenclature of Organic Chemistry were introduced to the CCOM program for 3D fragment searching. First, the search for the highest priority atom was limited to a distance of two bonds from the center bond for dihedral angle determination. Second, for indistinguishable atoms in experimentally observed solution structures, the smallest number of atom index in the molecular model was chosen as the priority atom for dihedral angle determination. A search of the 3D conformational fragment mb_3a6c4c of mevastatin () in combination with the SMiles ARbitrary Target Specification (SMARTS) description suggested that a change in the conformation of this fragment may be the driving force for dissociation of mevastatin from its target protein. Chirality 28:370-375, 2016. © 2016 Wiley Periodicals, Inc.
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Lovastatina/análogos & derivados , Conformação Molecular , Software , Lovastatina/química , Estrutura MolecularRESUMO
Inorganic nanomaterials endowed with hierarchical chirality could open new horizons in physical theory and applications because of their fascinating properties. Here, we report chiral ZnO films coated on quartz substrates with a hierarchical nanostructure ranging from atomic to micrometer scale. Three levels of hierarchical chirality exist in the ZnO films: helical ZnO crystalline structures that form primary helically coiled nanoplates, secondary helical stacking of these nanoplates, and tertiary nanoscale circinate aggregates formed by several stacked nanoplates. These films exhibited optical activity (OA) at 380â nm and in the range of 200-800â nm and created circularly polarized luminescence centered at 510â nm and Raman OA at 50-1400â cm(-1) , which was attributed to electronic transitions, scattering, photoluminescent emission, and Raman scattering in a dissymmetric electric field. The unprecedented strong OA could be attributed to multiple light scattering and absorption-enhanced light harvesting in the hierarchical structures.
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The unique enhanced sensitivity of vibrational circular dichroism (VCD) to the formation and development of amyloid fibrils in solution is extended to four additional fibril-forming proteins or peptides where it is shown that the sign of the fibril VCD pattern correlates with the sense of supramolecular filament chirality and, without exception, to the dominant fibril morphology as observed in AFM or SEM images. Previously for insulin, it has been demonstrated that the sign of the VCD band pattern from filament chirality can be controlled by adjusting the pH of the incubating solution, above pH 2 for "normal" left-hand-helical filaments and below pH 2 for "reversed" right-hand-helical filaments. From AFM or SEM images, left-helical filaments form multifilament braids of left-twisted fibrils while the right-helical filaments form parallel filament rows of fibrils with a flat tape-like morphology, the two major classes of fibril morphology that from deep UV resonance Raman scattering exhibit the same cross-ß-core secondary structure. Here we investigate whether fibril supramolecular chirality is the underlying cause of the major morphology differences in all amyloid fibrils by showing that the morphology (twisted versus flat) of fibrils of lysozyme, apo-α-lactalbumin, HET-s (218-289) prion, and a short polypeptide fragment of transthyretin, TTR (105-115), directly correlates to their supramolecular chirality as revealed by VCD. The result is strong evidence that the chiral supramolecular organization of filaments is the principal underlying cause of the morphological heterogeneity of amyloid fibrils. Because fibril morphology is linked to cell toxicity, the chirality of amyloid aggregates should be explored in the widely used in vitro models of amyloid-associated diseases.
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Amiloide/química , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Muramidase/química , Estrutura Secundária de Proteína , Estereoisomerismo , VibraçãoRESUMO
Characterization and understanding of protein higher order structure (HOS) is essential at all stages of biologics development. Here, two folding variants of a bispecific monoclonal antibody, the correctly folded form and an alternative configuration with reduced potency, were characterized by several HOS characterization techniques. Specifically, differential scanning calorimetry (DSC), circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), Raman and Raman optical activity (ROA) spectroscopy were used together to elucidate the impacts of disulfide bond scrambling in the fused scFv domains on the structure and thermal stability of the antibody. This study illustrates the importance of selecting appropriate biophysical characterization techniques based on the nature and magnitude of the HOS change.
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Anticorpos Biespecíficos , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Dissulfetos , Dissulfetos/química , Anticorpos Biespecíficos/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Estabilidade Proteica , Dobramento de Proteína , Análise Espectral Raman/métodos , Anticorpos Monoclonais/química , Conformação ProteicaRESUMO
Candida auris infection has been recognized as an urgent threat to antifungal drug resistance, and the Eagle effect of C. auris FKS1 (1,3-ß-d-glucan synthase) wild-type isolates has also been noted. The Eagle effect, namely, where higher concentrations of antifungals reduce fungicidal activity relative to lower concentrations, is a confounding factor of apparent antifungal resistance, but the detailed mechanism remains unclear. Here, we present the conformational variability of mutation sites for ERG11p (lanosterol 14α-demethylase) and FKS1 from deep neural network-based prediction along with the reported X-ray crystallographic and cryo-electron microscopy (cryo-EM) structures of antifungals. The sequence variability maps provide valuable insights into the inconsistent correlation between azole resistance and the mysterious Eagle effect with the dispersion of minimal inhibitory concentration (MIC) for echinocandin resistance. The conformational variability prediction supports the hypothesis that mutations K143R of clade I, VF125AL of clade III, and Y132F of clade IV for C. auris ERG11p make the corresponding site variable and that an increased population of invisible variable conformations potentially contributes to triazole resistance. In contrast, the predicted rigid conformation by the S639F mutation of hot spot region 1 (HS1) for FKS1 suggests that caspofungin (CAS) is involved in an uncompetitive inhibition, and a decreased population of the CAS-bound state of FKS1 with Rho1 leads to drug resistance. The predicted variable HS1 region for FKS1 WT isolates and the rigid one for FKS1 S639F mutants support the in vivo drug response and the in vitro MIC dispersion. A plausible mechanism of the Eagle effect is hereby proposed, namely, that a high concentration of CAS with a high membrane affinity reduces the population of the CAS-bound state of FKS1 with Rho1, as well as accompanying events such as aggregation or association depending on the conformational variability of HS1.
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The diastereomeric spiroiminodihydantoin-2'-deoxyribonucleoside (dSp) lesions resulting from 2'-deoxyguanosine (dG) or 8-oxo-7,8-dihydro-2'-deoxyguanosine (dOG) oxidation have generated much attention due to their highly mutagenic nature. Their propeller-like shape leads these molecules to display mutational profiles in vivo that are stereochemically dependent. However, there exist conflicting absolute configuration assignments arising from electronic circular dichroism (ECD) and NOESY-NMR experiments; thus, providing definitive assignments of the 3D structure of these molecules is of great interest. In the present body of work, we present data inconsistent with the reported ECD assignments for the dSp diastereomers in the nucleoside context, in which the first eluting isomer from a Hypercarb HPLC column was assigned to be the S configuration, and the second was assigned the R configuration. The following experiments were conducted: (1) determination of the diastereomer ratio of dSp products upon one-electron oxidation of dG in chiral hybrid or propeller G-quadruplexes that expose the re or si face to solvent, respectively; (2) absolute configuration analysis using vibrational circular dichroism (VCD) spectroscopy; (3) reinterpretation of the ECD experimental spectra using time-dependent density functional theory (TDDFT) with the inclusion of 12 explicit H-bonding waters around the Sp free bases; and (4) reevaluation of calculated specific rotations for the Sp enantiomers using the hydration model in the TDDFT calculations. These new insights provide a fresh look at the absolute configuration assignments of the dSp diastereomers in which the first eluting from a Hypercarb-HPLC column is (-)-(R)-dSp and the second is (+)-(S)-dSp. These assignments now provide the basis for understanding the biological significance of the stereochemical dependence of enzymes that process this form of DNA damage.
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DNA/química , Guanosina/análogos & derivados , Compostos de Espiro/química , 8-Hidroxi-2'-Desoxiguanosina , Dicroísmo Circular , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Elétrons , Quadruplex G , Guanosina/química , Modelos Moleculares , Conformação Molecular , EstereoisomerismoRESUMO
It is shown that fuzzy search and data mining techniques of supersecondary structure homology for subunits of proteins using conformational code patterns of α-helix-type (3ß5α4ß) and ß-sheet-type (6α4ß4ß) fragments can be used to extract correlations between fragments of MHC class I molecules and the light chain of immunoglobulins. The new method of conformational pattern analysis with fuzzy search of structural code homology reflects well the shape of main chain rather than secondary structure in comparison with the DSSP method. Further, the data mining technique using the combination of h- and s-fragment patterns can quantify the supersecondary structure homology between any subunits of proteins with different amino acid sequences. Characteristic fragment patterns (string "shhshss"), which were sandwiched between two identical amino acid sequences His and Pro, were found in light chains of various types of immunogloblins, α-chain and ß-2 microglobulin of MHC class I and α-chain and ß-chain of MHC class II, but not in heavy chains of Fab immunoglobulin fragments and T cell receptors (TCR). Leukocyte immunoglobulin-like receptors (LILR) are related by the conformational fragment (string "shhshss") to ß-2 microglobulins as a type of pair forms (string "sohsss"). Further, human IgM rheumatoid factor, one of the immunogloblins, did not strongly exhibit the conformational fragment pattern. Nonclassic MHC class I molecules CD1D, MIC-A, and MIC-B, which have functions to activate NKT, NK, and T cells, did not also clearly show the patterns. These code-driven mining techniques can be utilized as a metadata-generating tool for systems biology to elucidate the biological function of such conformational fragments of MHC I and II molecules, which come in contact with various signal ligands on the surface of T cells and natural killer cells.
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Mineração de Dados/métodos , Imunoglobulina M/química , Imunoglobulinas/química , Complexo Principal de Histocompatibilidade , Fator Reumatoide/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Lógica Fuzzy , Humanos , Imunoglobulina G/química , Cadeias Leves de Imunoglobulina/química , Camundongos , Modelos Moleculares , Fragmentos de Peptídeos/química , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/efeitos dos fármacosRESUMO
The Omicron BA.1 variant of SARS-CoV-2 preferentially infects through the cathepsin-mediated endocytic pathway, but the mechanism of cell entry has not been solved yet because BA.4/5 is more fusogenic and more efficiently spread in human lung cells than BA.2. It has been unclear why the Omicron spike is inefficiently cleaved in virions compared with Delta, and how the relatively effective reproduction proceeds without the cell entry through plasma membrane fusion. Conformational variability from deep neural network-based prediction correlates well with the thermodynamic stability of variants. The difference of seasonable pandemic variants in summer and those in winter is distinguishable by this conformational stability, and the geographical optimization of variants is also traceable. Further, the predicted conformational variability maps rationalize the less efficient S1/S2 cleavage of Omicron variants and provide a valuable insight into the cell entry through the endocytic pathway. It is concluded that conformational variability prediction is able to complement transformation information on motifs in protein structures for drug discovery.
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Fibrils are ß-sheet-rich aggregates that are generally composed of several protofibrils and may adopt variable morphologies, such as twisted ribbons or flat-like sheets. This polymorphism is observed for many different amyloid associated proteins and polypeptides. In a previous study we proposed the existence of another level of amyloid polymorphism, namely, that associated with fibril supramolecular chirality. Two chiral polymorphs of insulin, which can be controllably grown by means of small pH variations, exhibit opposite signs of vibrational circular dichroism (VCD) spectra. Herein, using atomic force microscopy (AFM) and scanning electron microscopy (SEM), we demonstrate that indeed VCD supramolecular chirality is correlated not only by the apparent fibril handedness but also by the sense of supramolecular chirality from a deeper level of chiral organization at the protofilament level of fibril structure. Our microscopic examination indicates that normal VCD fibrils have a left-handed twist, whereas reversed VCD fibrils are flat-like aggregates with no obvious helical twist as imaged by atomic force microscopy or scanning electron microscopy. A scheme is proposed consistent with observed data that features a dynamic equilibrium controlled by pH at the protofilament level between left- and right-twist fibril structures with distinctly different aggregation pathways for left- and right-twisted protofilaments.
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Insulina/química , Multimerização Proteica , Vibração , Animais , Bovinos , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína , EstereoisomerismoRESUMO
Spectral comparison is an important part of the assignment of the absolute configuration (AC) by vibrational circular dichroism (VCD), or equally by Raman optical activity (ROA). In order to avoid bias caused by personal interpretation, numerical methods have been developed to compare measured and calculated spectra. Using a neighbourhood similarity measure, the agreement between a computed and measured VCD or ROA spectrum is expressed numerically to introduce a novel confidence level measure. This allows users of vibrational optical activity (VOA) techniques (VCD and ROA) to assess the reliability of their assignment of the AC of a compound. To that end, a database of successful AC determinations is compiled along with neighbourhood similarity values between the experimental spectrum and computed spectra for both enantiomers. For any new AC determination, the neighbourhood similarities between the experimental spectrum and the computed spectra for both enantiomers are projected on the database allowing an interpretation of the reliability of their assignment.
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Algoritmos , Dicroísmo Circular , Conformação Molecular , Análise Espectral Raman , EstereoisomerismoRESUMO
Identifying the fundamental cause of transmissibility of multiple mutation strains and vaccine nullification is difficult in general and is a source of significant concern. The conformational variability of the mutation sites for B.1.617.2 (Δ), B.1.617.1 (κ), B.1.427/429 (ε), P.1 (γ), B.1.351 (ß), B.1.1.7 (α), S477N, and the wild-type strain has been assessed using a deep neural-network-based prediction program of conformational flexibility or rigidity in proteins (SSSCPreds). We find that although the conformation of G614 is rigid, which is assigned as a left-handed (LH) α-helix-type one, that of D614 is flexible without the hydrogen bonding latch to T859. The rigidity of glycine, which stabilizes the local conformation more effectively than that of aspartic acid with the latch, thereby contributes to the reduction of S1 shedding, high expression, and increase in infectivity. The finding that the sequence flexibility/rigidity map pattern of B.1.1.7 is similar to that of the wild-type strain but is largely different from those of B.1.351 and P.1 correlates with the minor escape ability of B.1.1.7. The increased rigidity of the amino acid sequence YRYRLFR from the SSSCPreds data of B.1.427/429 near the L452R mutation site contributes to the 2-fold increased B.1.427/B.1.429 viral shedding in vivo and the increase in transmissibility relative to wild-type circulating strains in a similar manner to D614G. The concordance and rigidity ratios of multiple mutation strains such as B.1.617.2 against the wild-type one at the receptor-binding domain (RBD) and receptor-binding motif (RBM) regions provide a good indication of the transmissibility and neutralization escape ability except for binding affinity of mutation sites such as N501Y.
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Amino acid mutations that improve protein stability and rigidity can accompany increases in binding affinity. Therefore, conserved amino acids located on a protein surface may be successfully targeted by antibodies. The quantitative deep mutational scanning approach is an excellent technique to understand viral evolution, and the obtained data can be utilized to develop a vaccine. However, the application of the approach to all of the proteins in general is difficult in terms of cost. To address this need, we report the construction of a deep neural network-based program for sequence-based prediction of supersecondary structure codes (SSSCs), called SSSCPrediction (SSSCPred). Further, to predict conformational flexibility or rigidity in proteins, a comparison program called SSSCPreds that consists of three deep neural network-based prediction systems (SSSCPred, SSSCPred100, and SSSCPred200) has also been developed. Using our algorithms we calculated here shows the degree of flexibility for the receptor-binding motif of SARS-CoV-2 spike protein and the rigidity of the unique motif (SSSC: SSSHSSHHHH) at the S2 subunit and has a value independent of the X-ray and Cryo-EM structures. The fact that the sequence flexibility/rigidity map of SARS-CoV-2 RBD resembles the sequence-to-phenotype maps of ACE2-binding affinity and expression, which were experimentally obtained by deep mutational scanning, suggests that the identical SSSC sequences among the ones predicted by three deep neural network-based systems correlate well with the sequences with both lower ACE2-binding affinity and lower expression. The combined analysis of predicted and observed SSSCs with keyword-tagged datasets would be helpful in understanding the structural correlation to the examined system.
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Aspongdopamines A and B (1 and 2), unusual adducts composed of N-acetyldopamine and adenine were isolated from the insect Aspongopus chinensis. Compounds 1 and 2 are positional isomers both isolated as racemates. Chiral separation assisted by 14-step total synthesis and computation including vibrational circular dichroism calculations allowed us to unambiguously assign the absolute configurations of eight stereoisomers. Renal fibrosis inhibition of the stereoisomers was evaluated in TGF-ß1-induced rat kidney epithelial cells.
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Adenina/síntese química , Produtos Biológicos/farmacologia , Dopamina/análogos & derivados , Insetos/efeitos dos fármacos , Fator de Crescimento Transformador beta1/química , Adenina/química , Animais , Dicroísmo Circular , Dopamina/síntese química , Dopamina/química , Estrutura Molecular , Ratos , Estereoisomerismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
A revised conformational code for the exhaustive analysis of conformers of all classes of molecules is proposed and applied to the vibrational circular dichroism (VCD) analysis of (S)-ibuprofen. The revised code can strictly define the conformation of compounds with relatively high-symmetry substituents and is especially useful for visualizing conformational changes in ligands and proteins. The conformational analysis of (S)-ibuprofen using the code in the solution state reveals that the four energetically preferred conformations, ibut-3alpha2alpha sigma(phpa-3alpha sigma2alpha), ibut-3alpha2alpha tau(phpa-3alpha tau2alpha), ibut-2beta3beta tau(phpa-3alpha tau2alpha), and ibut-2beta3beta sigma(phpa-3alpha sigma2alpha), exist in the monomer and dimer forms. In CDCl(3) solution, the dimer form is stabilized as the "U"-shape, and the ease of crystallization is largely ascribed to the conformation of phpa-3alpha2alpha for (S)-ibuprofen. The new version of the conformational code has the possibility to be used as a tool for the exhaustive analysis of conformers of all kinds of chemical compounds, conformome analysis, and in the future for metabolome, proteome, and genome analyses.
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Ibuprofeno/química , Dicroísmo Circular , Modelos Moleculares , Conformação Molecular , Ofloxacino/química , Espectrofotometria Infravermelho , Terminologia como AssuntoRESUMO
The absolute configuration and solution conformation of (R)-(+)-malathion were determined by using vibrational circular dichroism spectroscopy and a fragment-conformational search with a recently published conformational code. The determination of molecular stereochemistry was carried out without a conformational search using molecular mechanics calculations. Density functional theory calculations of the fragments of (R)-malathion, ethyl propionate, (R)-ethyl 2-(methylthio)propanoate, (R)-diethyl 2-(methylthio)succinate, and O,O,S-trimethyl phosphorodithioate were carried out, and the principal conformational features of the fragments were profiled. This fragment-conformational search reduces the time needed for the selection of the predominant conformations for (R)-malathion and significantly improves the accuracy of the determination of absolute configuration.
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Malation/química , Praguicidas/química , Dicroísmo Circular , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Soluções Farmacêuticas , Espectrofotometria Infravermelho , Estereoisomerismo , Termodinâmica , VibraçãoRESUMO
Measurements of near-infrared scattered circular polarization Raman optical activity (SCP-ROA) are presented using laser excitation at 780 nm for samples of S-(-)-alpha-pinene and L-alanyl-L-alanine. These are the first measurements of ROA outside the blue-to-green visible region between 488 and 532 nm. Comparison of Raman and ROA intensities measured with excitation at 532 and 780 nm demonstrate that the expected frequency to the fourth-power dependence for Raman scattering and the corresponding fifth-power dependence for ROA are observed. It can be concluded that, to within this frequency dependence, the same level of efficiency of Raman and ROA measurements using commercial instrumentation with 532 nm excitation is maintained with the change to near-infrared excitation at 780 nm.
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Espectrofotometria Infravermelho/métodos , Análise Espectral Raman/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We report the first vibrational circular dichroism (VCD) measurement of spatial heterogeneity in a sample using infrared (IR) microsampling. Vibrational circular dichroism spectra are typically measured using a standard IR cell with an IR beam diameter of 10 mm or greater making it impossible to investigate the spatial heterogeneity of a solid film sample. We have constructed a VCD sampling assembly with either 3 mm or 1 mm spatial resolution. An XY-translation stage was used to measure spectra at different spatial locations producing IR and VCD maps of the sample. In addition, a rotating sample stage was employed using a dual photoelastic modulator (PEM) setup to suppress artifacts due to linear birefringence in solid-phase or film samples. Infrared and VCD mapping of an insulin fibril film has been carried out at both 3 and 1 mm spatial resolution, and lysozyme films were mapped at 1 mm resolution. The IR spectra of different spots vary in intensity due primarily to sample thickness. The changes in the VCD intensity across the map largely correlate to corresponding changes in the IR map. Closer inspection of the insulin map revealed changes in the relative intensities of the VCD spectra not present in the parent IR spectra, which indicated differences in the degree of supramolecular chirality of the fibrils in the various spatial regions. For lysozyme films, in addition to different degrees of supramolecular chirality, reversal of the net fibril chirality was observed. The large signal-to-noise ratio observed at 1 mm resolution implies the feasibility of further increasing the spatial resolution by one or two orders of magnitude for protein fibril film samples.
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Amiloide/análise , Amiloide/química , Dicroísmo Circular/métodos , Animais , Artefatos , Bovinos , Insulina/análise , Insulina/química , Muramidase/análise , Muramidase/química , Processamento de Sinais Assistido por Computador , Espectroscopia de Infravermelho com Transformada de Fourier , VibraçãoRESUMO
The first use of near-infrared (NIR) Fourier transform vibrational circular dichroism (FT-VCD) to follow changes in the enantiomeric excess (EE) of chiral sample molecules in time using a flow-cell sampling apparatus is reported. Simultaneous changes in the fractional composition and the EE of a mixture of two different chiral molecules were monitored as a function of time. This simulates the progress of the chemical reaction from a chiral reactant to a chiral product where the mole fractions and EE values of both species may change with time. For the molecules studied, alpha-pinene, camphor, and borneol, the accuracy of following EE changes for one species alone is approximately 2%, while for simultaneously following EE changes in two species it is approximately 3% for 30 min sampling periods at 16 cm(-1) spectral resolution. These findings demonstrate the potential for VCD to be used in the NIR region for real-time monitoring of the composition and %EE of chemical reactions involving the synthesis of chiral molecules.