Chiral electromagnetic fields generated by arrays of nanoslits.

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.

Ultrasensitive detection and characterization of biomolecules using superchiral fields.

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 ß-sheet content, which suggests that this approach could form the basis for assaying technologies capable of detecting amyloid diseases and certain types of viruses.

Time reversal and molecular properties.

Chemists regularly exploit point group symmetry in their analyses of molecular structure and properties but they rarely utilize time reversal symmetry. The time reversal operator T reverses the momenta and spins of all particles in a system and distinguishes properties which are even under T, such as the electric dipole moment, from those that are odd, such as the magnetic dipole moment. We review the role of T in quantum mechanics and discuss its application to the properties of molecules in electric and magnetic fields. Among the properties considered are natural and magnetic optical activity, magneto-chiral effects, antisymmetric Raman scattering, optical NMR and ESR, chirality, and absolute enantioselection.

Solution structure of native proteins with irregular folds from Raman optical activity.

Raman optical activity (ROA) spectra have been measured for the proteins hen phosvitin, yeast invertase, bovine alpha-casein, soybean Bowman-Birk protease inhibitor, and rabbit Cd(7)-metallothionein, all of which have irregular folds in the native state. The results show that ROA is able to distinguish between two types of disorder. Specifically, invertase, alpha-casein, the Bowman-Birk inhibitor, and metallothionein appear to possess a "static" type of disorder similar to that in disordered states of poly(L-lysine) and poly(L-glutamic acid); whereas phosvitin appears to possess a more "dynamic" type of disorder similar to that in reduced (unfolded) lysozyme and ribonuclease A and also in molten globule protein states. In the delimiting cases, static disorder corresponds to that found in loops and turns within native proteins with well-defined tertiary folds that contain sequences of residues with fixed but nonrepetitive phi,psi angles; and dynamic disorder corresponds to that envisaged for the model random coil in which there is a distribution of Ramachandran phi,psi angles for each amino acid residue, giving rise to an ensemble of interconverting conformers. In both cases there is a propensity for the phi,psi angles to correspond to the alpha, beta and poly(L-proline) II (PPII) regions of the Ramachandran surface, as in native proteins with well-defined tertiary folds. Our results suggest that, with the exception of invertase and metallothionein, an important conformational element present in the polypeptide and protein states supporting the static type of disorder is that of the PPII helix. Long sequences of relatively unconstrained PPII helix, as in alpha-casein, may impart a plastic (rheomorphic) character to the structure.

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.

Raman optical activity characterization of native and molten globule states of equine lysozyme: comparison with hen lysozyme and bovine alpha-lactalbumin.

Vibrational Raman optical activity (ROA) spectra of the calcium-binding lysozyme from equine milk in native and nonnative states are measured and compared with those of the homologous proteins hen egg white lysozyme and bovine alpha-lactalbumin. The ROA spectrum of holo equine lysozyme at pH 4.6 and 22 degrees C closely resembles that of hen lysozyme in regions sensitive to backbone and side chain conformations, indicating similarity of the overall secondary and tertiary structures. However, the intensity of a strong positive ROA band at approximately 1340 cm(-1), which is assigned to a hydrated form of alpha helix, is more similar to that in the ROA spectrum of bovine alpha-lactalbumin than hen lysozyme and may be associated with the greater flexibility and calcium-binding ability of equine lysozyme and bovine alpha-lactalbumin compared with hen lysozyme. In place of a strong sharp positive ROA band at approximately 1300 cm(-1) in hen lysozyme that is assigned to an alpha helix in a more hydrophobic environment, equine lysozyme shows a broader band centered at approximately 1305 cm(-1), which may reflect greater heterogeneity in some alpha-helical sequences. The ROA spectrum of apo equine lysozyme at pH 4.6 and 22 degrees C is almost identical to that of the holo protein, which indicates that loss of calcium has little influence on the backbone and side chain conformations, including the calcium-binding loop. From the similarity of their ROA spectra, the A state at pH 1.9 and both 2 and 22 degrees C and the apo form at pH 4.5 and 48 degrees C, which are partially folded denatured (molten globule or state A) forms of equine lysozyme, have similar structures that the ROA suggests contain much hydrated alpha helix. The A state of equine lysozyme is shown by these results to be more highly ordered than that of bovine alpha-lactalbumin, the ROA spectrum of which has more features characteristic of disordered states. A positive tryptophan ROA band at approximately 1551 cm(-1) in the native holo protein disappears in the A state, which is probably due to the presence of nonnative conformations of the tryptophans associated with a previously identified cluster of hydrophobic residues.

Solution structure and dynamics of biomolecules from Raman optical activity.

Raman optical activity (ROA) measures vibrational optical activity by means of a small difference in the intensity of Raman scattering from chiral molecules in right and left circularly polarized incident laser light. The ROA spectra of a wide range of biomolecules in aqueous solution can now be measured routinely. Because of its sensitivity to the chiral elements of biomolecular structure, ROA provides new information about solution structure and dynamics complementary to that supplied by conventional spectroscopic techniques. This article provides a brief introduction to the theory and practice of ROA spectroscopy followed by a review of recent ROA results on polypeptides, proteins, carbohydrates, nucleic acids and viruses which illustrate how new insight into current problems of structure, folding and function may be obtained from ROA studies.

Raman optical activity of filamentous bacteriophages: hydration of alpha-helices.

We report the first observations of vibrational Raman optical activity (ROA) on intact viruses. Specifically, ROA spectra of the filamentous bacteriophages Pf1, M13 and IKe in aqueous solution were measured in the range approximately 600-1800 cm-1. On account of its ability to probe directly the chiral elements of biomolecular structure, ROA has provided a new perspective on the solution structures of these well-studied systems. The ROA spectra of all three are dominated by signatures of helical elements in the major coat proteins, as expected from pre-existing data. The helical elements generate strong sharp positive ROA bands at approximately 1300 and 1342 cm-1in H2O solution, but in2H2O solution the approximately 1342 cm-1bands disappear completely. The spectra are similar to those of polypeptides under conditions that produce alpha-helical conformations. Our present results, together with results from other studies, suggest that the positive approximately 1342 cm-1ROA bands are generated by a highly hydrated form of alpha-helix, and that the positive approximately 1300 cm-1bands originate in alpha-helix in a more hydrophobic environment. The presence of significant amounts of highly hydrated helical sequences accords with the known flexibility of these viruses. Differences of spectral detail for Pf1, M13 and IKe demonstrate that ROA is sensitive to subtle variations of conformation and hydration within the major coat proteins, with M13 and IKe possibly containing more non-helical structure than Pf1. The ROA spectra of Pf1 at temperatures above and below that at which a structural transition is known to occur (approximately 10 degrees C) reveal little difference in the protein conformation between the two forms, but there are indications of changes in DNA structure.

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: an incisive new probe of the structure and dynamics of biomolecules.

Determination of the solution structure and function of biomolecules such as proteins, carbohydrates and nucleic acids remains at the forefront of biomedical research. Thanks to recent developments in instrumentation, biomolecules can now be studied using Raman optical activity (ROA), a novel technique which measures vibrational optical activity in chiral molecules by means of a small difference in the intensity of Raman scattering in right- and left-circularly polarized incident laser light. This article reviews recent progress in the application of ROA to the determination of the solution structure and dynamics of proteins, carbohydrates and nucleic acids.

Evidence for global mobility in the premelting of a polynucleotide from temperature-dependent Raman optical activity.

The backscattered Raman and Raman optical activity (ROA) spectra of poly(rA)-poly(rU) at 20 degrees C and 45 degrees C in buffered aqueous solution between 650 and 1750 cm(-1) are reported. Although the intensity of the majority of the Raman bands increase by varying amounts as the temperature is raised in accordance with the well-known hypochromic effect, the reverse effect is found for the ROA signals which we attribute to thermal accessibility of a greater number of distinct conformations leading to cancellation of ROA signals. The difference ROA spectrum obtained by subtracting the spectrum recorded at 45 degrees C from that recorded at 20 degrees C displays a very similar sign pattern to those at both 20 degrees C and 45 degrees C throughout the spectral region examined. This indicates that the same average structure is maintained in this temperature range and that the thermal fluctuations are correlated through the bases, the glycosidic link, the sugar ring, and the phosphate backbone of both strands. These results indicate that ROA may be a useful new probe of the dynamics of nucleic acid in solution.

The lubricant of life: a proposal that solvent water promotes extremely fast conformational fluctuations in mobile heteropolypeptide structure.

Recent observations using the novel technique of Raman optical activity suggest that individual residues in unfolded proteins and in disordered loop regions of molten globule-like states cluster in the alpha-helix, beta-structure, and PPII-helix regions of the Ramachandran surface and that they "flicker" between these regions at rates approximately 10(12) s-1 at room temperature. It is proposed that these rapid motions, which occur on the same picosecond time scale as rearrangements of the hydrogen bond network in bulk water, are promoted by solvent water molecules via a repertoire of transient hydrated reverse turn conformations. Some implications of this proposal for protein folding and function are discussed.

Homochirality as the signature of life: the SETH Cigar.

A characteristic hallmark of life is its homochirality: all biomolecules are usually of one hand, e.g. on Earth life uses only L-amino acids for protein synthesis and not their D mirror images. It is therefore suggested that a search for extra-terrestrial life can be approached as a Search for Extra-Terrestrial Homochirality (SETH). A novel miniaturized space polarimeter, called the SETH Cigar, is described which could he used to detect optical rotation as the homochiral signature of life on other planets. Moving parts are avoided by replacing the normal rotating polarizer by multiple fixed polarizers at different angles as in the eye of the bee. It is believed that homochirality will be found in the subsurface layers on Mars as a relic of extinct life.

Residual structure in unfolded proteins revealed by Raman optical activity.

Because of its ability to probe directly the chiral elements of the peptide backbone, together with the very short time scale of the scattering process, vibrational Raman optical activity (ROA) can provide new information on structure in non-native states of proteins. Here we report ROA studies of hen egg white lysozyme and bovine ribonuclease A in unfolded denatured states, prepared by reducing all the disulfide bonds. ROA spectra of unfolded lysozyme at 45, 20, and 2 degrees C, and of unfolded ribonuclease A at 35 and 20 degrees C, are presented and discussed. At 45 and 20 degrees C, unfolded lysozyme appears to contain very little extended secondary structure, but at 2 degrees C there could be roughly 20% of the native amount of alpha-helix present but little beta-sheet. Unfolded ribonuclease A, on the other hand, appears to contain roughly 50% of its native-like secondary structure, including both alpha-helix and beta-sheet, at 20 degrees C; similar secondary structure persists at 35 degrees C, but the amount is reduced. The most striking result is the observation of three sharp ROA bands in the extended amide III region, originating in coupled C alpha-H and N-H deformations, which might monitor directly the dominant intrinsic propensities for residues to adopt particular phi, psi angles, averaged over the different amino acids in the mobile heteropolypeptide. Specifically, positive bands at approximately 1300 and 1314 cm-1 appear to monitor propensities for alpha-helix and beta-structure, respectively, and a negative band at approximately 1237 cm-1 appears to monitor that for the poly(L-proline) II helix. These signals are generated by individual residues clustering in the most favorable regions of the Ramachandran plot and are present even in the absence of signals from the corresponding extended secondary structures. At 45 degrees C, the 1300 and 1314 cm-1 ROA bands of unfolded lysozyme coalesce into a single sharp band from which an analysis similar to that used for exchange effects in NMR suggests a rate of approximately 2.6 x 10(12) s-1 for interconversion between the individual residue conformations at this temperature.

The native-like tertiary fold in molten globule alpha-lactalbumin appears to be controlled by a continuous phase transition.

On account of its ability to discriminate between secondary, loop and sidegroup structure and its special sensitivity to conformational mobility, vibrational Raman optical activity (ROA) has provided new insights into the complexity of order within the molten globule state from measurements on alpha-lactalbumin at pH 2.0 over the temperature range 2 to 45 degrees C. Thus while much of the secondary structure present in the native protein persists with only a small gradual decrease with increasing temperature, the tertiary backbone fold changes dramatically, being almost complete and native-like at 2 degrees C and almost completely disordered at 35 degrees C. The change of the tertiary fold with temperature is cooperative but has no latent heat, and so has the approximate characteristics of a continuous phase transition, being of the order-disorder type since it involves the interconversion of rigid, locally-ordered loop structure with disordered mobile backbone structure. This has implications for protein folding because the long-range correlations that exist in the critical region of a continuous (but not in a first-order) phase transition could resolve, in principle, the problem of how the protein finds its native-like folding pattern at the molten globule stage.

Vibrational Raman optical activity of alpha-lactalbumin: comparison with lysozyme, and evidence for native tertiary folds in molten globule states.

Proteins in aqueous solution are now accessible to Raman optical activity (ROA) measurements, which provide an incisive new probe of secondary and tertiary structure illustrated here by a study of bovine alpha-lactalbumin. The room-temperature ROA spectrum of native bovine alpha-lactalbumin is similar to that of native hen egg-white lysozyme except for features attributable to differences in the loop regions: in particular, a positive ROA band at approximately 1338 cm-1 assigned to conformationally homogeneous loop structure, possibly with local order corresponding to 3(10)-helix, has more than double the intensity in alpha-lactalbumin compared with lysozyme. This is consistent with the two proteins having similar secondary structure but different local details in the tertiary fold. ROA measurements on alpha-lactalbumin at pH 2.0 over a range of temperatures have provided a new perspective on the molten globule state. Thus at 35 degrees C ROA reveals the presence of some secondary structure but an almost complete loss of the tertiary loop structure; whereas at 2 degrees C the ROA spectrum is almost identical with that of the native protein, which is strong evidence that virtually all of the secondary structure and the tertiary backbone fold persist, albeit within a looser framework associated with increased solvent exposure and change of environment of many of the side-chains as evidenced by an increase in noise and bandwidth of some of the ROA signals together with aromatic fluorescence and near-UV circular dichroism signals characteristic of the molten globule state. Our sample of acid alpha-lactalbumin at 2 degrees C therefore appears to be an archetypal example of Ptitsyn's "native-like" molten globule, having a fixed native-like tertiary fold but with loss of tight packing of the side-chains; whereas at 35 degrees C it is a "disordered" molten globule. At 20 degrees C the acid molten globule appears to retain highly native-like secondary structure but with most of the tertiary fold already lost. A calcium-free sample of alpha-lactalbumin at neutral pH displayed a broad cooperative transition between native and molten globule states at approximately 15 degrees C, with the latter state showing similar but somewhat degraded tertiary loop ROA signatures to the native protein. In both the acid and apo molten globule states the ROA signatures of the secondary structure and the tertiary loops showed a gradual change with temperature.