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Following its first observation 50 years ago Raman optical activity (ROA), which refers to a circular polarization dependence of Raman scattering from chiral molecules, has evolved into a powerful chiroptical spectroscopy for studying a large range of biomolecules in aqueous solution. Among other things ROA provides information about motif and fold as well as secondary structure of proteins; structure of carbohydrates and nucleic acids; polypeptide and carbohydrate structure of intact glycoproteins; and protein and nucleic acid structure of intact viruses. Quantum chemical simulations of observed Raman optical activity spectra can provide complete three-dimensional structures of biomolecules, together with information about conformational dynamics. This article reviews how ROA has provided new insight into the structure of unfolded/disordered states and sequences, ranging from the complete disorder of the random coil to the more controlled type of disorder exemplified by poly L-proline II helix in proteins, high mannose glycan chains in glycoproteins and constrained dynamic states of nucleic acids. Possible roles for this 'careful disorderliness' in biomolecular function, misfunction and disease are discussed, especially amyloid fibril formation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Optical spectroscopy can be used to quickly characterise the structural properties of individual molecules. However, it cannot be applied to biological assemblies because light is generally blind to the spatial distribution of the component molecules. This insensitivity arises from the mismatch in length scales between the assemblies (a few tens of nm) and the wavelength of light required to excite chromophores (≥150 nm). Consequently, with conventional spectroscopy, ordered assemblies, such as the icosahedral capsids of viruses, appear to be indistinguishable isotropic spherical objects. This limits potential routes to rapid high-throughput portable detection appropriate for point-of-care diagnostics. Here, we demonstrate that chiral electromagnetic (EM) near fields, which have both enhanced chiral asymmetry (referred to as superchirality) and subwavelength spatial localisation (∼10 nm), can detect the icosahedral structure of virus capsids. Thus, they can detect both the presence and relative orientation of a bound virus capsid. To illustrate the potential uses of the exquisite structural sensitivity of subwavelength superchiral fields, we have used them to successfully detect virus particles in the complex milieu of blood serum.

Manipulating symmetry environments of metal ions to control functional properties is a fundamental concept of chemistry. For example, lattice strain enables control of symmetry in solids through a change in the nuclear positions surrounding a metal centre. Light-matter interactions can also induce strain but providing dynamic symmetry control is restricted to specific materials under intense laser illumination. Here, we show how effective chemical symmetry can be tuned by creating a symmetry-breaking rotational bulk polarisation in the electronic charge distribution surrounding a metal centre, which we term a meta-crystal field. The effect arises from an interface-mediated transfer of optical spin from a chiral light beam to produce an electronic torque that replicates the effect of strain created by high pressures. Since the phenomenon does not rely on a physical rearrangement of nuclear positions, material constraints are lifted, thus providing a generic and fully reversible method of manipulating effective symmetry in solids.

Protein-protein interactions (PPIs) play a pivotal role in many biological processes. Discriminating functionally important well-defined protein-protein complexes formed by specific interactions from random aggregates produced by nonspecific interactions is therefore a critical capability. While there are many techniques which enable rapid screening of binding affinities in PPIs, there is no generic spectroscopic phenomenon which provides rapid characterization of the structure of protein-protein complexes. In this study we show that chiral plasmonic fields probe the structural order and hence the level of PPI specificity in a model antibody-antigen system. Using surface-immobilized Fab' fragments of polyclonal rabbit IgG antibodies with high specificity for bovine serum albumin (BSA), we show that chiral plasmonic fields can discriminate between a structurally anisotropic ensemble of BSA-Fab' complexes and random ovalbumin (OVA)-Fab' aggregates, demonstrating their potential as the basis of a useful proteomic technology for the initial rapid high-throughput screening of PPIs.

Structural analysis of carbohydrates is a complicated endeavour, due to the complexity and diversity of the samples at hand. Herein, we apply a combined computational and experimental approach, employing molecular dynamics (MD) and density functional theory (DFT) calculations together with NMR and Raman optical activity (ROA) measurements, in the structural study of three mannobiose disaccharides, consisting of two mannoses with varying glycosidic linkages. The disaccharide structures make up the scaffold of high mannose glycans and are therefore important targets for structural analysis. Based on the MD population analysis and NMR, the major conformers of each mannobiose were identified and used as input for DFT analysis. By systematically varying the solvent models used to describe water interacting with the molecules and applying overlap integral analysis to the resulting calculational ROA spectra, we found that a full quantum mechanical/molecular mechanical approach is required for an optimal calculation of the ROA parameters. Subsequent normal mode analysis of the predicted vibrational modes was attempted in order to identify possible marker bands for glycosidic linkages. However, the normal mode vibrations of the mannobioses are completely delocalised, presumably due to conformational flexibility in these compounds, rendering the identification of isolated marker bands unfeasible., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The structural order of biopolymers, such as proteins, at interfaces defines the physical and chemical interactions of biological systems with their surroundings and is hence a critical parameter in a range of biological problems. Known spectroscopic methods for routine rapid monitoring of structural order in biolayers are generally only applied to model single-component systems that possess a spectral fingerprint which is highly sensitive to orientation. This spectroscopic behavior is not a generic property and may require the addition of a label. Importantly, such techniques cannot readily be applied to real multicomponent biolayers, have ill-defined or unknown compositions, and have complex spectroscopic signatures with many overlapping bands. Here, we demonstrate the sensitivity of plasmonic fields with enhanced chirality, a property referred to as superchirality, to global orientational order within both simple model and "real" complex protein layers. The sensitivity to structural order is derived from the capability of superchiral fields to detect the anisotropic nature of electric dipole-magnetic dipole response of the layer; this is validated by numerical simulations. As a model study, the evolution of orientational order with increasing surface density in layers of the antibody immunoglobulin G was monitored. As an exemplar of greater complexity, superchiral fields are demonstrated, without knowledge of exact composition, to be able to monitor how qualitative changes in composition alter the structural order of protein layers formed from blood serum, thereby establishing the efficacy of the phenomenon as a tool for studying complex biological interfaces.

The structure adopted by biomaterials, such as proteins, at interfaces is a crucial parameter in a range of important biological problems. It is a critical property in defining the functionality of cell/bacterial membranes and biofilms (i.e., in antibiotic-resistant infections) and the exploitation of immobilized enzymes in biocatalysis. The intrinsically small quantities of materials at interfaces precludes the application of conventional spectroscopic phenomena routinely used for (bio)structural analysis due to a lack of sensitivity. We show that the interaction of proteins with superchiral fields induces asymmetric changes in retardation phase effects of excited bright and dark modes of a chiral plasmonic nanostructure. Phase retardations are obtained by a simple procedure, which involves fitting the line shape of resonances in the reflectance spectra. These interference effects provide fingerprints that are an incisive probe of the structure of interfacial biomolecules. Using these fingerprints, layers composed of structurally related proteins with differing geometries can be discriminated. Thus, we demonstrate a powerful tool for the bioanalytical toolbox.

In the past few decades, Raman optical activity (ROA) spectroscopy has been shown to be very sensitive to the solution structure of peptides and proteins. A major and urgent challenge remains the need to make detailed assignments of experimental ROA patterns and relate those to the solution structure adopted by the protein. In the past few years, theoretical developments and implementations of ROA theory have made it possible to use quantum chemical methods to compute the ROA spectra of peptides. In this work, a large database of ROA spectra of peptide model structures describing the allowed backbone conformations of proteins was systematically calculated and used to make unprecedented detailed assignments of experimental ROA patterns to the conformational elements of the peptide in solution. By using a similarity index to compare an experimental spectrum to the database spectra (2902 theoretical spectra), the conformational preference of the peptide in solution can be assigned to a very specific region in the Ramachandran space. For six (poly)peptides this approach was validated and gives excellent agreement between experiment and theory. Additionally, hydrogen/deuterium exchanged structures and the conformational dependence of the amide modes in Raman spectra can be analysed using the new database. The excellent agreement between experiment and theory demonstrates the power of the newly developed database as a tool to study Raman and ROA patterns of peptides and proteins. The interpretation of experimental ROA patterns of different proteins published in the scientific literature is discussed based on the spectral trends observed in the database.

The refractive index sensitivity of plasmonic fields has been exploited for over 20 years in analytical technologies. While this sensitivity can be used to achieve attomole detection levels, they are in essence binary measurements that sense the presence/absence of a predetermined analyte. Using plasmonic fields, not to sense effective refractive indices but to provide more "granular" information about the structural characteristics of a medium, provides a more information rich output, which affords opportunities to create new powerful and flexible sensing technologies not limited by the need to synthesize chemical recognition elements. Here we report a new plasmonic phenomenon that is sensitive to the biomacromolecular structure without relying on measuring effective refractive indices. Chiral biomaterials mediate the hybridization of electric and magnetic modes of a chiral solid-inverse plasmonic structure, resulting in a measurable change in both reflectivity and chiroptical properties. The phenomenon originates from the electric-dipole-magnetic-dipole response of the biomaterial and is hence sensitive to biomacromolecular secondary structure providing unique fingerprints of α-helical, β-sheet, and disordered motifs. The phenomenon can be observed for subchiral plasmonic fields (i.e., fields with a lower chiral asymmetry than circularly polarized light) hence lifting constraints to engineer structures that produce fields with enhanced chirality, thus providing greater flexibility in nanostructure design. To demonstrate the efficacy of the phenomenon, we have detected and characterized picogram quantities of simple model helical biopolymers and more complex real proteins.

Optimal performance of nanophotonic devices, including sensors and solar cells, requires maximizing the interaction between light and matter. This efficiency is optimized when active moieties are localized in areas where electromagnetic (EM) fields are confined. Confinement of matter in these 'hotspots' has previously been accomplished through inefficient 'top-down' methods. Here we report a rapid 'bottom-up' approach to functionalize selective regions of plasmonic nanostructures that uses nano-localized heating of the surrounding water induced by pulsed laser irradiation. This localized heating is exploited in a chemical protection/deprotection strategy to allow selective regions of a nanostructure to be chemically modified. As an exemplar, we use the strategy to enhance the biosensing capabilities of a chiral plasmonic substrate. This novel spatially selective functionalization strategy provides new opportunities for efficient high-throughput control of chemistry on the nanoscale over macroscopic areas for device fabrication.

Development of low-cost disposable plasmonic substrates is vital for the applicability of plasmonic sensing. Such devices can be made using injection-molded templates to create plasmonic films. The elements of these plasmonic films are hybrid nanostructures composed of inverse and solid structures. Tuning the modal coupling between the two allows optimization of the optical properties for nanophotonic applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Optical spectroscopic methods do not routinely provide information on higher order hierarchical structure (tertiary/quaternary) of biological macromolecules and assemblies. This necessitates the use of time-consuming and material intensive techniques, such as protein crystallography, NMR, and electron microscopy. Here we demonstrate a spectroscopic phenomenon, superchiral polarimetry, which can rapidly characterize ligand-induced changes in protein higher order (tertiary/quaternary) structure at the picogram level, which is undetectable using conventional CD spectroscopy. This is achieved by utilizing the enhanced sensitivity of superchiral evanescent fields to mesoscale chiral structure.

We demonstrate that engineered artificial gold chiral nanostructures display significant levels of non-linear optical activity even without plasmonic enhancement. Our work suggests that although plasmonic excitation enhances the intensity of second harmonic emission it is not a prerequisite for significant non-linear (second harmonic) optical activity. It is also shown that the non-linear optical activities of both the chiral nanostructures and simple chiral molecules on surfaces have a common origin, namely pure electric dipole excitation. This is a surprising observation given the significant difference in length scales, three orders of magnitude, between the nanostructures and simple chiral molecules. Intuitively, given that the dimensions of the nanostructures are comparable to the wavelength of visible light, one would expect non-localised higher multipole excitation (e.g. electric quadrupole and magnetic dipole) to make the dominant contribution to non-linear optical activity. This study provides experimental evidence that the electric dipole origin of non-linear optical activity is a generic phenomenon which is not limited to sub-wavelength molecules and assemblies. Our work suggests that viewing non-plasmonic nanostructures as "meta-molecules" could be useful for rationally designing substrates for optimal non-linear optical activity.

The discrete symmetries of parity P, time reversal T, and charge conjugation C may be used to characterize the properties of chiral systems. It is well known that parity violation infiltrates into ordinary matter via an interaction between the nucleons and electrons, mediated by the Z(0) particle, that lifts the degeneracy of the mirror-image enantiomers of a chiral molecule. Being odd under P but even under T, this P-violating interaction exhibits true chirality and so may induce absolute enantioselection under all circumstances. It has been suggested that CP violation may also infiltrate into ordinary matter via a P-odd, T-odd interaction mediated by the (as yet undetected) axion. This CP-violating interaction exhibits false chirality and so may induce absolute enantioselection in processes far from equilibrium. Both true and false cosmic chirality should be considered together as possible sources of homochirality in the molecules of life., (Copyright © 2012 Wiley Periodicals, Inc.)

A selection of my work on chirality is sketched in two distinct parts of this lecture. Symmetry and Chirality explains how the discrete symmetries of parity P, time reversal T, and charge conjugation C may be used to characterize the properties of chiral systems. The concepts of true chirality (time-invariant enantiomorphism) and false chirality (time-noninvariant enantiomorphism) that emerge provide an extension of Lord Kelvin's original definition of chirality to situations where motion is an essential ingredient thereby clarifying, inter alia, the nature of physical influences able to induce absolute enantioselection. Consideration of symmetry violations reveals that strict enantiomers (exactly degenerate) are interconverted by the combined CP operation. Raman optical activity surveys work, from first observation to current applications, on a new chiroptical spectroscopy that measures vibrational optical activity via Raman scattering of circularly polarized light. Raman optical activity provides incisive information ranging from absolute configuration and complete solution structure of smaller chiral molecules and oligomers to protein and nucleic acid structure of intact viruses., (Copyright © 2012 Wiley Periodicals, Inc., A Wiley Company.)

Using a modal matching theory, we demonstrate the generation of short-range, chiral electromagnetic fields via the excitation of arrays of staggered nanoslits that are chiral in two dimensions. The electromagnetic near fields, which exhibit a chiral density greater than that of circularly polarized light, can enhance the chiroptical interactions in the vicinity of the nanoslits. We discuss the features of nanostructure symmetry required to obtain the chiral fields and explicitly show how these structures can give rise to detection and characterization of materials with chiral symmetry.

Splitting it up: excellent agreement between the experimental and the quantum-chemically simulated Raman optical activity (ROA) spectrum of (+)-poly(trityl methacrylate) shows that the polymer backbone adopts a left-handed helical conformation while the trityl side groups display a left-handed propeller conformation. Thus ROA can be used to determine the complete structure of synthetic chiral polymers in solution., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The spectroscopic analysis of large biomolecules is important in applications such as biomedical diagnostics and pathogen detection, and spectroscopic techniques can detect such molecules at the nanogram level or lower. However, spectroscopic techniques have not been able to probe the structure of large biomolecules with similar levels of sensitivity. Here, we show that superchiral electromagnetic fields, generated by the optical excitation of plasmonic planar chiral metamaterials, are highly sensitive probes of chiral supramolecular structure. The differences in the effective refractive indices of chiral samples exposed to left- and right-handed superchiral fields are found to be up to 10(6) times greater than those observed in optical polarimetry measurements, thus allowing picogram quantities of adsorbed molecules to be characterized. The largest differences are observed for biomolecules that have chiral planar sheets, such as proteins with high β-sheet content, which suggests that this approach could form the basis for assaying technologies capable of detecting amyloid diseases and certain types of viruses.

Polyproline II (PPII) helix is an extended secondary structure present in a number of proteins. PPII-containing sequences mediate specific protein-protein interactions with partners containing appropriate cognate domains called PPII-recognizing domains (PRDs) and are involved in the activation of intracellular signaling pathways. Thus, the identification of PPII structures in proteins is of great interest, not only to explore molecular and physiological mechanisms, but also to elaborate new potential drugs. By revisiting X-ray crystal structures of liganded alpha-type human estrogen receptor (ERalpha), we have identified an 11-residue PPII-helical sequence (D(321)AEPPILYSEY(331)) in the ligand-binding domain of the receptor. The data recorded by far-ultraviolet circular dichroism (far-UV CD), vibrational Raman optical activity (ROA) and differential scanning calorimetry (DSC) show that the corresponding peptide (Ac-DAEPPILYSEY-NH(2)) is particularly well structured in PPII, with the same proportion of PPII as observed from X-ray structures (approximately 85%). In addition, studies carried out on ERalpha-negative Evsa-T breast cancer cells transiently co-transfected with a pcDNA3-ERalpha plasmid and a Vit-tk-Luc reporter gene revealed that the peptide antagonizes the estradiol-induced transcription providing perspectives for researching new molecules with antagonistic properties.

Vibrational Raman optical activity (ROA), measured as small circularly polarized components in Raman scattering from chiral molecules, was applied to study the backbone conformations of the first five generations of poly(L-lysine) dendrigrafts (DGLs) in water. Generation 1 was found to support predominantly the poly(L-proline) II (PPII) conformation, the amount of which steadily decreased with increasing generation, with a concomitant increase in other backbone conformations. This behavior may be due to increasing crowding of the lysine side chains, together with suppression of backbone hydration, with increasing branching. In contrast, the ROA spectra of a series of linear poly(L-lysine)s in water show little change with increasing molecular weight. Our results may have implications for the nonimmunogenic properties of DGLs.

The samples used for the first observations of vibrational Raman optical activity (ROA) in 1972, namely both enantiomers of 1-phenylethanol and 1-phenylethylamine, have been revisited using a modern commercial ROA instrument together with state-of-the-art ab initio calculations. The simulated ROA spectra reveal for the first time the vibrational origins of the first reported ROA signals, which comprised similar couplets in the alcohol and amine in the spectral range approximately 280-400 cm(-1). The results demonstrate how easy and routine ROA measurements have become, and how current ab initio quantum-chemical calculations are capable of simulating experimental ROA spectra quite closely provided sufficient averaging over accessible conformations is included. Assignment of absolute configuration is, inter alia, completely secure from results of this quality. Anharmonic corrections provided small improvements in the simulated Raman and ROA spectra. The importance of conformational averaging emphasized by this and previous related work provides the underlying theoretical background to ROA studies of dynamic aspects of chiral molecular and biomolecular structure and behavior., ((c) 2009 Wiley-Liss, Inc.)

A pair of enantiomerically pure quaternary ammonium salts with a chiral side chain, methyl-(R)-(1-methylpropyl)di(n-propyl)ammonium iodide 1 and methyl-(S)-(1-methylpropyl)di(n-propyl)ammonium iodide 2, and the related racemate, methyl-(rac)-(1-methylpropyl)di(n-propyl)ammonium iodide 3, were synthesized through a reductive alkylation procedure, starting from enantiomerically pure and, also, racemic forms of (rac)-(1-methylpropyl)amine. A spectroscopic chiroptical signature in solution was provided by the Raman optical activity spectra of compounds 1 and 2. The crystallographic structures of 1, 2, and 3 were examined by single crystal X-ray diffraction. 1 crystallizes in the tetragonal space group P4(3)2(1)2 (no. 96), a = b = 12.826 (2) A, c = 17.730 (2) A, V = 2916.9 (5) A(3), Z = 8, Flack coefficient 0.04 (2). 2 crystallizes in the tetragonal space group P4(1)2(1)2 (no. 92), a = b = 12.842 (1) A, c = 17.749 (2) A, V = 2927.0 (5) A(3), Z = 8, Flack coefficient 0.05 (2). The crystal structures and space groups for 1 and 2 are enantiomorphs and the crystallographic investigation confirmed the absolute configuration of the stereocenter in both compounds. 3 crystallizes in the monoclinic space group P2(1)/n(no. 14), a = 8.178 (1) A, b = 14.309 (2) A, c = 12.328 (2) A, beta = 96.811 (6) degrees, V = 1432.4 (2) A(3), Z = 4., (2008 Wiley-Liss, Inc.)

The binding of divalent copper ions to the full-length recombinant murine prion protein PrP23-231 at neutral pH was studied using vibrational Raman optical activity (ROA) and ultraviolet circular dichroism (UV CD). The effect of the Cu2+ ions on PrP structure depends on whether they are added after refolding of the protein in water or are present during the refolding process. In the first case ROA reveals that the hydrated alpha-helix is lost, with UV CD revealing a drop from approximately 25% to approximately 18% in the total alpha-helix content. The lost alpha-helix could be that comprising residues 145-156, located within the region associated with scrapie PrP formation. In the second case, ROA reveals the protein's structure to be almost completely disordered/irregular, with UV CD revealing a drop in total alpha-helix content to approximately 5%. Hence, although Cu2+ binding takes place exclusively within the unfolded/disordered N-terminal region, it can profoundly affect the structure of the folded/alpha-helical C-terminal region. This is supported by the finding that refolding in the presence of Cu2+ of a mutant in which the first six histidines associated with copper binding to the N-terminal region are replaced by alanine has a similar alpha-helix content to the metal-free protein. In contrast, when the protein is refolded in the presence of divalent manganese ions, ROA indicates the alpha-helix is reinforced, with UV CD revealing an increase in total alpha-helix content to approximately 30%. The very different influence of Cu2+ and Mn2+ ions on prion protein structure may originate in the different stability constants and geometries of their complexes.

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., ((c) 2007 Wiley-Liss, Inc.)

Rich and complex Raman scattering and Raman optical activity (ROA) spectra have been measured monitoring the pH induced alpha-helix-to-disordered conformational transition in poly(L-glutamic acid). Two-dimensional (2D) correlation techniques have been applied to facilitate a comprehensive analysis of these two complementary spectral sets. Synchronous contour plots have identified band assignments of alpha-helical and disordered conformations, and have revealed bands characteristic of changes in the protonation state of the polypeptide. Asynchronous plots, on the other hand, have probed the relative sequential orders of intensity changes indicating a decrease in intensity of alpha-helical bands in the backbone skeletal stretch region, followed by a subsequent decrease in intensity in the extended amide III and amide I regions, underlying the appearance of disordered structure, including poly(L-proline) II (PPII) helix. The application of a 2D correlation 'moving' window has also disclosed two distinct phases during helix unfolding in the alpha-helix-to-disordered transition, occurring at approximately pH 4.9 and approximately pH 5.2, possibly a result of the difference in helical stability between the end and central regions of the alpha-helix. This paper demonstrates the potential value of combining 2D Raman, 2D ROA and moving window correlation techniques for the detailed investigation of complex and subtle changes of secondary structure during the unfolding mechanisms of polypeptides and proteins.

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.

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.

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., ((c) 2005 Wiley-Liss, Inc.)

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.

A vibrational Raman optical activity (ROA) study of bovine alpha1-acid glycoprotein (AGP) is reported. Using the recently introduced ChiralRAMAN instrument from BioTools, Inc., a high-quality ROA spectrum of AGP, measured as a small circularly polarized component in the scattered light, was obtained in the range of 200-1800 cm-1. Comparison with the ROA spectra of beta-lactoglobulin and N,N'-diacetylchitobiose reveals features consistent with previous suggestions that the peptide component of AGP has a structure based on the lipocalin fold, and that the first two glycosidic links after the N-links to asparagine in the pentasaccharide core are of the beta(1-4)-type. A detailed analysis of the band patterns may ultimately provide information on the more conformationally heterogeneous and functionally crucial peripheral oligosaccharide segments. Hence, information about both the polypeptide and carbohydrate components may be obtained from the ROA spectra of intact glycoproteins.

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.

We synthesized by solution methods a water-soluble, terminally blocked heptapeptide based on five markedly helicogenic, C(alpha)-tetrasubstituted alpha-amino acids C(alpha)-methyl-L-norvalines and two strongly hydrophilic 2-amino-3-[1-(1,4,7-triazacyclononane)]-L-propanoic acid residues at positions 2 and 5. A Fourier transform infrared absorption and NMR analysis in deuterated chloroform and aqueous solutions of the heptapeptide and two side-chain protected synthetic precursors confirmed our working hypothesis that all oligomers are folded in the 3(10)-helical conformation. Based on these findings, we exploited this heptapeptide as a chiral reference compound for detailed electronic CD, vibrational CD, and Raman optical activity characterizations of the 3(10)-helix in aqueous solution.

A vibrational Raman optical activity (ROA) study, supplemented by protein X-ray crystal structure data, of alpha-helices in polypeptides, proteins, and viruses has suggested that ROA bands in the extended amide III spectral region may be used to distinguish between two types of right-handed alpha-helix. One type, associated with a positive ROA band at approximately 1300 cm(-1), dominates in hydrophobic environments and appears to be unhydrated; the other, associated with a positive ROA band at approximately 1340 cm(-1), dominates in hydrophilic environments and appears to be hydrated. Evidence is presented to support the hypothesis that unhydrated alpha-helix corresponds to the canonical conformation alpha(c) and hydrated alpha-helix to a more open conformation alpha(o) stabilized by hydrogen bonding of a water molecule or a hydrophilic side chain to the peptide carbonyl. Alpha-helical poly(L-lysine) and poly(L-ornithine) in aqueous solution and poly(L-alanine) in dichloracetic acid display both bands, but alpha-helical poly(l-glutamic acid) in aqueous solution and poly(gamma-benzyl L-glutamate) in CHCl(3) display only the approximately 1340 cm(-1) band and so may exist purely as alpha(o) due to enhanced stabilization of this conformation by particular side chain characteristics. The ROA spectrum of poly(beta-benzyl L-aspartate) in CHCl(3) reveals that it exists in a single left-handed alpha-helical state more analogous to alpha(o) than to alpha(c).

A vibrational Raman optical activity (ROA) study of a series of alanine peptides in aqueous solution is presented. The seven-alanine peptide Acetyl-OOAAAAAAAOO-Amide (OAO), recently shown by NMR and UVCD to adopt a predominantly poly(l-proline II) (PPII) helical conformation in aqueous solution, gave an ROA spectrum very similar to that of disordered poly(l-glutamic acid) which has long been considered to adopt the PPII conformation, both being dominated by a strong positive extended amide III ROA band at approximately 1319 cm-1 together with weak positive amide I ROA intensity at approximately 1675 cm-1. A series of alanine peptides Ala2-Ala6 studied in their cationic states in aqueous solution at low pH displayed ROA spectra which steadily evolved toward that of OAO with increasing chain length. As well as confirming that alanine peptides can support the PPII conformation in aqueous solution, our results also confirm the previous ROA band assignments for PPII structure, thereby reinforcing the foundation for ongoing ROA studies of unfolded and partially folded proteins.

The vibrational Raman optical activity (ROA) spectrum of a polypeptide in a model beta-sheet conformation, that of poly(l-lysine), was measured for the first time, and the alpha-helix --> beta-sheet transition monitored as a function of temperature in H(2)O and D(2)O. Although no significant population of a disordered backbone state was detected at intermediate temperatures, some side chain bands not present in either the alpha-helix or beta-sheet state were observed. The observation of ROA bands in the extended amide III region assigned to beta-turns suggests that, under our experimental conditions, beta-sheet poly(L-lysine) contains up-and-down antiparallel beta-sheets based on the hairpin motif. The ROA spectrum of beta-sheet poly(L-lysine) was compared with ROA data on a number of native proteins containing different types of beta-sheet. Amide I and amide II ROA band patterns observed in beta-sheet poly(L-lysine) are different from those observed in typical beta-sheet proteins and may be characteristic of an extended flat multistranded beta-sheet, which is unlike the more irregular and twisted beta-sheet found in most proteins. However, a reduced isoform of the truncated ovine prion protein PrP(94-233) that is rich in beta-sheet shows amide I and amide II ROA bands similar to those of beta-sheet poly(L-lysine), which suggests that the C-terminal domain of the prion protein is able to support unusually flat beta-sheets. A principal component analysis (PCA) that identifies protein structural types from ROA band patterns provides a useful representation of the structural relationships among the polypeptide and protein states considered in the study.

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.

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.

A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity to chirality, ROA is an incisive probe of their aqueous solution structures at the molecular level. Protein ROA bands are especially prominent from which, as we have shown by comparison with the ROA spectra of proteins with known structures and by using a pattern recognition program, the folds of the major coat protein subunits may be deduced. Information about amino acid side-chain conformations, exemplified here by the determination of the sign and magnitude of the torsion angle chi(2,1) for tryptophan in fd, may also sometimes be obtained. By subtracting the ROA spectrum of the empty protein capsid (top component) of cowpea mosaic virus from those of the intact middle and bottom-upper components separated by means of a caesium chloride density gradient, the ROA spectrum of the viral RNA was obtained, which revealed that the RNA takes up an A-type single-stranded helical conformation and that the RNA conformations in the middle and bottom-upper components are very similar. This information is not available from the X-ray crystal structure of cowpea mosaic virus since no nucleic acid is visible.

Potato virus X (PVX) and narcissus mosaic virus (NMV) were studied using vibrational Raman optical activity (ROA) in order to obtain new information on the structures of their coat protein subunits. The ROA spectra of the two intact virions are very similar to each other and similar to that of tobacco mosaic virus (TMV) studied previously, being dominated by signals characteristic of proteins with helix bundle folds. In particular, PVX and NMV show strong positive ROA bands at approximately 1340 cm(-1) assigned to hydrated alpha-helix and perhaps originating in surface exposed helical residues, together with less strong positive ROA intensity in the range approximately 1297-1312 cm(-1) assigned to alpha-helix in a more hydrophobic environment and perhaps originating in residues at helix-helix interfaces. The positive approximately 1340 cm(-1) ROA band of TMV is less intense than those of PVX and NMV, suggesting that TMV contains less hydrated alpha-helix. Small differences in other spectral regions reflect differences in some loop, turn and side-chain compositions and conformations among the three viruses. A pattern recognition program based on principal component analysis of ROA spectra indicates that the coat protein subunit folds of PVX and NMV may be very similar to each other and similar to that of TMV. These results suggest that PVX and NMV may have coat protein subunit structures based on folds similar to the TMV helix bundle and hence that the helical architecture of the PVX and NMV particles may be similar to that of TMV but with different structural parameters.

The casein milk proteins and the brain proteins alpha-synuclein and tau have been described as natively unfolded with random coil structures, which, in the case of alpha-synuclein and tau, have a propensity to form the fibrils found in a number of neurodegenerative diseases. New insight into the structures of these proteins has been provided by a Raman optical activity study, supplemented with differential scanning calorimetry, of bovine beta- and kappa-casein, recombinant human alpha-, beta- and gamma-synuclein, together with the A30P and A53T mutants of alpha-synuclein associated with familial cases of Parkinson's disease, and recombinant human tau 46 together with the tau 46 P301L mutant associated with inherited frontotemporal dementia. The Raman optical activity spectra of all these proteins are very similar, being dominated by a strong positive band centred at approximately 1318 cm(-1) that may be due to the poly(l-proline) II (PPII) helical conformation. There are no Raman optical activity bands characteristic of extended secondary structure, although some unassociated beta strand may be present. Differential scanning calorimetry revealed no thermal transitions for these proteins in the range 15-110 degrees C, suggesting that the structures are loose and noncooperative. As it is extended, flexible, lacks intrachain hydrogen bonds and is hydrated in aqueous solution, PPII helix may impart a rheomorphic (flowing shape) character to the structure of these proteins that could be essential for their native function but which may, in the case of alpha-synuclein and tau, result in a propensity for pathological fibril formation due to particular residue properties.