Chiral Sulfur Compounds Studied by Raman Optical Activity: tert-Butanesulfinamide and its Precursor tert-Butyl tert-Butanethiosulfinate

Two chiral sulfur compounds, tert-butyl tert-butanethiosulfinate (1) and tert-butanesulfinamide (2), with inversion of configuration, have been studied by Raman optical activity (ROA) and electronic circular dichroism combined with density functional theory calculation. With the S-S linkage in 1, the couplings between the two tertiary carbon atoms often generate large ROA signals, whereas the tertiary carbon atom itself generally makes a large contribution to ROA signals in 2 for similar vibrational modes. The conformational dependence of ROA parameters provides probing conformation around the S-S bond from a new perspective. The simultaneous use of electronic circular dichroism and ROA is warranted to extract reliable conformational information. ROA provides a suitable candidate for the stereochemical study of chiral sulfur compounds, especially its capability of sensing the conformation around the S-S bond. Chirality 24:731-740, 2012. ? 2012 Wiley Periodicals, Inc.     

Vibrational Raman optical activity of proteins,nucleic acids,and viruses

Due to its sensitivity to chirality, Raman optical activity (ROA), which may be measured as a small difference in vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light, is a powerful probe of biomolecular structure in solution. Protein ROA spectra provide information on the secondary and tertiary structures of the polypeptide backbone, hydration, side-chain conformation, and structural elements present in denatured states. Nucleic acid ROA spectra yield information on the sugar ring conformation, the base stacking arrangement, and the mutual orientation of the sugar and base rings around the C-N glycosidic linkage. ROA is able to simultaneously probe the structures of both the protein and the nucleic acid components of intact viruses. This article gives a brief account of the theory and measurement of ROA and presents the ROA spectra of a selection of proteins, nucleic acids, and viruses which illustrate the applications of ROA spectroscopy in biomolecular research.     

Spectroscopic properties of the nonplanar amide group: a computational study

Experimental studies suggest that amide bond may significantly deviate from planar arrangement even in linear peptides and proteins. In order to find out the extent to which such deviation may influence principal amide spectroscopic properties, we conducted a computational study of nonplanar N-methylacetamide (NMA) conformers. Vibrational absorption, Raman, and electronic spectra including optical activity were simulated with ab initio and density functional theory (DFT) methods. According to the results, small nonplanarity deviations may be detectable by nonpolarized spectroscopic techniques, albeit as subtle spectral changes. The optical activity methods, such as the vibrational circular dichroism (VCD), Raman optical activity (ROA), and electronic circular dichroism (CD, ECD), provide enhanced information about the amide nonplanarity, because planar amide is not optically active (chiral). For VCD, however, the inherently chiral contribution in most peptides and proteins most probably provides very weak signal in comparison with other contributions, such as the dipolar coupling. For the electronic CD, the nonplanarity contribution is relatively big and causes a strong CD couplet in the n-pi* absorption region accompanied by a red frequency shift. The pi-pi* CD region is relatively unaffected. The ROA spectroscopy appears most promising for the nonplanarity detection and the inherent chiral signal may dominate entire spectral parts. The amide I and III vibrational ROA bands are most challenging experimentally because of their relatively weak coupling to other peptide vibrations.     

Raman optical activity of proteins, carbohydrates and glycoproteins

On account of its sensitivity to chirality, Raman optical activity (ROA), which may be measured as a small difference in the intensity of vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the structure of biomolecules. Protein ROA spectra provide information on secondary and tertiary structures of polypeptide backbones, backbone hydration and side-chain conformations, and on structural elements present in unfolded states. Carbohydrate ROA spectra provide information on the central features of carbohydrate stereochemistry, especially that of the glycosidic link. Glycoprotein ROA spectra provide information on both the polypeptide and carbohydrate components. This article describes the ROA technique and presents and discusses the ROA spectra of a selection of proteins, carbohydrates, and a glycoprotein. The many structure-sensitive bands in protein ROA spectra are favorable for applying pattern recognition techniques, illustrated here using nonlinear mapping, to determine structural relationships between different proteins.     

Structure and behaviour of biomolecules from Raman optical activity

Raman optical activity, which can be measured as a small circularly polarized component in Raman-scattered light from chiral molecules, holds much promise for studying a large range of biomolecules in aqueous solution. Among other things, it provides information about motif and fold, as well as secondary structure, of proteins; the solution structure of carbohydrates; and the structure of the polypeptide and carbohydrate components of intact glycoproteins. In addition, new insights into the structural elements present in unfolded protein sequences, and the structure of the protein and nucleic acid components of intact viruses can be obtained. Ab initio quantum-chemical simulations of observed Raman optical activity spectra provide the complete three-dimensional structure of small biomolecules. Raman optical activity measurements are now routine thanks to the availability of a commercial instrument based on a novel design.     

Residual structure in disordered peptides and unfolded proteins from multivariate analysis and ab initio simulation of Raman optical activity data

Vibrational Raman optical activity (ROA), measured as a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the aqueous solution structure of proteins. The large number of structure-sensitive bands in protein ROA spectra makes multivariate analysis techniques such as nonlinear mapping (NLM) especially favorable for determining structural relationships between different proteins. We have previously used NLM to map a large dataset of peptide, protein, and virus ROA spectra into a readily visualizable two-dimensional space in which points close to or distant from each other, respectively, represent similar or dissimilar structures. As well as folded proteins, our dataset contains ROA spectra from many natively unfolded proteins, proteins containing both folded and unfolded domains, denatured partially structured molten globule and reduced protein states, together with folded proteins containing little or no alpha-helix or beta-sheet. In this article, the relative positions of these systems in the NLM plot are used to obtain information about any residual structure that they may contain. The striking differences between the structural propensities of proteins that are unfolded in their native states and those that are unfolded due to denaturation may be responsible for their often very different behavior, especially with regard to aggregation. An ab initio simulation of the Raman and ROA spectra of an alanine oligopeptide in the poly(L-proline) II-helical conformation confirms previous suggestions that this conformation is a significant structural element in disordered peptides and natively unfolded proteins. The use of ROA to identify and characterize proteins containing significant amounts of unfolded structure will, inter alia, be valuable in structural genomics/proteomics since unfolded sequences often inhibit crystallization.     

Raman and Raman optical activity (ROA) analysis of RNA structural motifs in Domain I of the EMCV IRES

Raman and Raman optical activity (ROA) spectra were collected for four RNA oligonucleotides based on the EMCV IRES Domain I to assess the contributions of helix, GNRA tetraloop, U·C mismatch base pair and pyrimidine-rich bulge structures to each. Both Raman and ROA spectra show overall similarities for all oligonucleotides, reflecting the presence of the same base paired helical regions and GNRA tetraloop in each. Specific bands are sensitive to the effect of the mismatch and asymmetric bulge on the structure of the RNA. Raman band changes are observed that reflect the structural contexts of adenine residues, disruption of A-form helical structure, and incorporation of pyrimidine bases in non-helical regions. The ROA spectra are also sensitive to conformational mobility of ribose sugars, and verify a decrease in A-type helix content upon introduction of the pyrimidine-rich bulge. Several Raman and ROA bands also clearly show cooperative effects between the mismatch and pyrimidine-rich bulge motifs on the structure of the RNA. The complementary nature of Raman and ROA spectra provides detailed and highly sensitive information about the local environments of bases, and secondary and tertiary structures, and has the potential to yield spectral signatures for a wide range of RNA structural motifs.     

Delineation of protein structure classes from multivariate analysis of protein Raman optical activity data

Vibrational Raman optical activity (ROA), measured as a small difference in the intensity of Raman scattering from chiral molecules in right and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the aqueous solution structure of proteins. On account of the large number of structure-sensitive bands in protein ROA spectra, multivariate analysis techniques such as non-linear mapping (NLM) are especially favourable for determining structural relationships between different proteins. Here NLM is used to map a dataset of 80 polypeptide, protein and virus ROA spectra, considered as points in a multidimensional space with axes representing the digitized wavenumbers, into readily visualizable two and three-dimensional spaces in which points close to or distant from each other, respectively, represent similar or dissimilar structures. Discrete clusters are observed which correspond to the seven structure classes all alpha, mainly alpha, alphabeta, mainly beta, all beta, mainly disordered/irregular and all disordered/irregular. The average standardised ROA spectra of the proteins falling within each structure class have distinct features characteristic of each class. A distinct cluster containing the wheat protein A-gliadin and the plant viruses potato virus X, narcissus mosaic virus, papaya mosaic virus and tobacco rattle virus, all of which appear in the mainly alpha cluster in the two-dimensional representation, becomes clearly separated in the direction of increasing disorder in the three-dimensional representation. This suggests that the corresponding five proteins, none of which to date has yielded high-resolution X-ray structures, consist mainly of alpha-helix and disordered structure with little or no beta-sheet. This combination of structural elements may have functional significance, such as facilitating disorder-to-order transitions (and vice versa) and suppressing aggregation, in these proteins and also in sequences within other proteins. The use of ROA to identify proteins containing significant amounts of disordered structure will, inter alia, be valuable in structural genomics/proteomics since disordered regions often inhibit crystallization.     

Electronic and vibrational optical activity of several peptides related to neurohypophyseal hormones: disulfide group conformation

Electronic and vibrational optical activity of the set of neurohypophyseal hormones and their analogs was investigated to clarify the S-S bond solution conformation. The selected compounds include oxytocin (I), lysine vasopressin (II), arginine vasopressin (III), and their analogs (IV-IX), differing widely in their pharmacological properties. We have extended the already known electronic circular dichroism data by new information provided by vibrational circular dichroism (VCD) and Raman optical activity (ROA). The use of VCD brought additional details on three-dimensional structure of the chain reversal in the ring moiety and on its left handedness. Furthermore, Raman scattering and ROA allowed us to deduce the sense of the disulfide bond torsion.     

New insight into the pH-dependent conformational changes in bovine beta-lactoglobulin from Raman optical activity.

We have studied the conformation of beta-lactoglobulin in aqueous solution at room temperature over the pH range approximately 2.0-9.0 using vibrational Raman optical activity (ROA). The ROA spectra clearly show that the basic up and down beta-barrel core is preserved over the entire pH range, in agreement with other studies. However, from the shift of a sharp positive ROA band at approximately 1268 to approximately 1294 cm(-1) on going from pH values below that of the Tanford transition, which is centered at pH approximately 7.5, to values above, the Tanford transition appears to be associated with changes in the local conformations of residues in loop sequences possibly corresponding to a migration into the alpha-helical region of the Ramachandran surface from a nearby region. These changes may be related to those detected in X-ray crystal structures which revealed that the Tanford transition is associated with conformational changes in loops which form a doorway to the interior of the protein. The results illustrate how the ability of ROA to detect loop and turn structure separately from secondary structure is useful for studying conformational plasticity in proteins.     

Raman optical activity: a tool for protein structure analysis

On account of its sensitivity to chirality, Raman optical activity (ROA), measured here as the intensity of a small, circularly polarized component in the scattered light using unpolarized incident light, is a powerful probe of protein structure and behavior. Protein ROA spectra provide information on secondary and tertiary structures of polypeptide backbones, backbone hydration, and side chain conformations, and on structural elements present in unfolded states. This article describes the ROA technique and presents ROA spectra, recorded with a commercial instrument of novel design, of a selection of proteins to demonstrate how ROA may be used to readily distinguish between the main classes of protein structure. A principal component analysis illustrates how the many structure-sensitive bands in protein ROA spectra are favorable for applying pattern recognition techniques to determine structural relationships between different proteins.     

Beta-sheet and associated turn signatures in vibrational Raman optical activity spectra of proteins.

We have measured the aqueous solution vibrational Raman optical activity (ROA) spectra of concanavalin A, alpha-chymotrypsin, and beta-lactoglobulin, all of which are rich in beta-sheet, together with that of the model beta-turn peptide L-pro-L-leu-gly-NH2. Possible ROA signatures of antiparallel beta-sheet include a strong sharp positive band at approximately 1,313 cm-1 associated with backbone amide III C alpha H and NH deformations, and an amide I couplet, negative at low wavenumber and positive at high, centered at approximately 1,658 cm-1. Negative ROA bands in the range approximately 1,340-1,380 cm-1, which might originate in glycine CH2 deformations, appear to be characteristic of beta-turns. Our results provide further evidence that ROA is a more incisive probe of protein conformation than conventional vibrational spectroscopy, infrared, or Raman, because only those few vibrational coordinates within a given normal mode that sample the skeletal chirality directly contribute to the corresponding ROA band intensity.     

New insight into the solution structures of wheat gluten proteins from Raman optical activity

Vibrational Raman optical activity (ROA) spectra of the wheat proteins alpha-gliadin (A-gliadin), omega-gliadin, and a 30 kDa peptide called T-A-1 from the high molecular weight glutenin subunit (HMW-GS) Dx5 were measured to obtain new information about their solution structures. The spectral data show that, under the conditions investigated, A-gliadin contains a considerable amount of hydrated alpha-helix, most of which probably lies within a relatively structured C-terminal domain. Smaller quantities of beta-structure and poly(l-proline) II (PPII) helix were also identified. Addition of methanol was found to increase the alpha-helix content at the expense of some of the beta and PPII structure. In comparison, omega-gliadin and the T-A-1 peptide were found to consist of large amounts of well-defined PPII structure with some turns but no alpha-helix. The results for the T-A-1 peptide are in agreement with a model in which HMW-GS are extended but not highly rigid. Application of a pattern recognition technique, based on principal component analysis (PCA), to the ROA spectra reinforces these conclusions.     

Raman optical activity study on insulin amyloid- and prefibril intermediate

The amyloid fibril of bovine insulin and its renaturing intermediates were studied by using Raman optical activity (ROA). In the spectrum of the amyloid, the sharp +/- ROA couplet of amide I band characteristic of the β-sheet-rich proteins was observed, together with a sharp peak at 1271 cm(-1) characteristic of a turn structure. The shoulder ROA peak of the native insulin at ~ 1340 cm(-1), which was assigned to the hydrated α-helix, was not observed in the amyloid, suggesting that the hydrated α-helix was converted to the parallel β-sheet structure in the amyloid. Recovery of the amyloid to the native state was also monitored by ROA. The intermediate states showed distinct features from the amyloid or native ones. The intermediates did not show a characteristic ROA peak of the poly(L-proline) II helix at ~ 1318 cm(-1). The hydrated α-helix ROA peak was not recovered in the intermediate states. In a process of the amyloid formation, at first the hydrated α-helix of the native insulin is converted to a specific partially unfolded structure, and then, it was converted to the parallel β-sheet structure with many turns.     

Is polyproline II helix the killer conformation? A Raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme

The amyloidogenic prefibrillar partially denatured intermediate of human lysozyme, prepared by heating the native protein to 57 degrees C at pH 2.0, was studied using Raman optical activity (ROA). A positive band in the room temperature ROA spectrum of the native protein at approximately 1345 cm(-1), assigned to a hydrated form of alpha-helix, is not present in that of the prefibrillar intermediate, where a new strong positive band at approximately 1318 cm(-1) appears instead that is assigned to the poly(l-proline) II (PPII)-helical conformation. A sharp negative band at approximately 1241 cm(-1) in the native protein, assigned to beta-strand, shows little change in the ROA spectrum of the prefibrillar intermediate. The disappearance of a positive ROA band at approximately 1551 cm(-1) assigned to vibrations of tryptophan side-chains indicates that major conformational changes have occurred among the five tryptophan residues present in human lysozyme, four of which are located in the alpha-domain. The various ROA data suggest that a substantial loss of tertiary structure has occurred in the prefibrillar intermediate and that this is located more in the alpha-domain than in the beta-domain. There is no evidence for any increase in beta-structure. The ROA spectrum of hen lysozyme, which does not form amyloid fibrils so readily, remains much more native-like on heating to 57 degrees C at pH 2.0. The thermal behaviour of the alanine-rich alpha-helical peptide AK21 in aqueous solution was found to be similar to that of human lysozyme. Hydrated alpha-helix therefore appears to readily undergo a conformational change to PPII structure on heating, which may be a key step in the conversion of alpha-helix into beta-sheet in the formation of amyloid fibrils in human lysozyme. Since it is extended, flexible, lacks intrachain hydrogen bonds and is fully hydrated in aqueous solution, PPII helix has the appropriate characteristics to be implicated as a critical conformational element in many conformational diseases. Disorder of the PPII type may be a sine qua non for the formation of regular fibrils; whereas the more dynamic disorder of the random coil may lead only to amorphous aggregates.     

Intramolecular Enantiomerism in S(+)2,2‐dimethyl ‐1,3‐dioxolane‐4‐methanol: The Interpretation of Raman Optical Activity Intensity

A bond polarizability algorithm was developed and applied to interpret the Raman optical activity (ROA) intensity. It is demonstrated that for the chiral molecule such as S(+)2,2‐dimethyl‐1,3‐dioxolane‐4‐methanol there exists approximate (or symmetry breaking) mirror reflection that reverses the signs of the differential bond polarizabilities of the pair bond coordinates that are related to each other by the mirror reflection, just like that between the right and left enantiomers. The magnitude difference of the differential bond polarizabilities of the pair bond coordinates becomes smaller as they are farther away from the asymmetric atom. Hence, that the asymmetric atom (center) plays a central role in ROA is confirmed from a spectroscopic viewpoint. Meanwhile, the concept of intramolecular enantiomerism is proposed. Chirality 27:820–825, 2015. © 2015 Wiley Periodicals, Inc.     

Determination of protein fold class from Raman or Raman optical activity spectra using random forests

Knowledge of the fold class of a protein is valuable because fold class gives an indication of protein function and evolution. Fold class can be accurately determined from a crystal structure or NMR structure, though these methods are expensive, time-consuming, and inapplicable to all proteins. In contrast, vibrational spectra [infra-red, Raman, or Raman optical activity (ROA)] are rapidly obtained for proteins under wide range of biological molecules under diverse experimental and physiological conditions. Here, we show that the fold class of a protein can be determined from Raman or ROA spectra by converting a spectrum into data of 10 cm⁻¹ bin widths and applying the random forest machine learning algorithm. Spectral data from 605 and 1785 cm⁻¹ were analyzed, as well as the amide I, II, and III regions in isolation and in combination. ROA amide II and III data gave the best performance, with 33 of 44 proteins assigned to one of the correct four top-level structural classification of proteins (SCOP) fold class (all α, all β, α and β, and disordered). The method also shows which spectral regions are most valuable in assigning fold class.     

The elongation and contraction of actin bundles are induced by double-headed myosins in a motor concentration-dependent manner

The aqueous solution structure of the full-length recombinant ovine prion protein PrP(25-233), together with that of the N-terminal truncated version PrP(94-233), have been studied using vibrational Raman optical activity (ROA) and ultraviolet circular dichroism (UVCD). A sharp positive band at approximately 1315 cm(-1) characteristic of poly(L-proline) II (PPII) helix that is present in the ROA spectrum of the full-length protein is absent from that of the truncated protein, together with bands characteristic of beta-turns. Although it is not possible similarly to identify PPII helix in the full-length protein directly from its UVCD spectrum, subtraction of the UVCD spectrum of PrP(94-233) from that of PrP(25-233) yields a difference UVCD spectrum also characteristic of PPII structure and very similar to the UVCD spectrum of murine PrP(25-113). These results provide confirmation that a major conformational element in the N-terminal region is PPII helix, but in addition show that the PPII structure is interspersed with beta-turns and that little PPII structure is present in PrP(94-233). A principal component analysis of the ROA data indicates that the alpha-helix and beta-sheet content, located in the structured C-terminal domain, of the full-length and truncated proteins are similar. The flexibility imparted by the high PPII content of the N-terminal domain region may be an essential factor in the function and possibly also the misfunction of prion proteins.     

Remote Excited Raman Optical Activity of Adenine Along Ag Plasmonic Waveguide

We report the remote excited Raman optical activity (ROA) of adenine along Ag plasmonic waveguide. First, the surface plasmons that propagate along Ag nanowire is demonstrated experimentally. Second, the Raman spectra of adenine are measured experimentally. Third, the remote exited ROA by plasmonic waveguide are measured and compared. It is found that the plasmon chirality strongly influenced the molecular ORA by the local surface plasmon and remote plasmon waveguide. The plasmon chirality of nanostructures and the chirality plasmon waveguide should be considered in the experiments for the local and remote measurement.