The past 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. The past years, theoretical developments and implementations of ROA theory have made it possible to use quantum chemical methods to compute ROA spectra of peptides. In this work, a large database of ROA spectra of peptide model structures describing the allowed backbone conformations of proteins were 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 scientific literature is discussed based on the spectral trends observed in the database.

High-resolution resonance Raman (RR) and resonance Raman optical activity (ROA) spectra of cytochrome c were obtained in order to perform full assignment of spectral features of the resonance ROA spectrum. The resonance ROA spectrum of cytochrome c revealed a distinct spectral signature pattern due to resonance enhanced skeletal porphyrin vibrations, more pronounced than any contribution from the protein backbone. Combining the intrinsic resonance properties of cytochrome c with the surface plasmon enhancement achieved with colloidal silver particles, the surface enhanced resonance Raman scattering (SERRS) and surface enhanced resonance ROA (SERROA) spectra of the protein were successfully obtained at concentrations as low as 1 microM. The assignments of spectral features were based on the information obtained from the RR and resonance ROA spectra. Excellent agreement between RR and SERRS spectra is reported, while some disparities were observed between the resonance ROA and SERROA spectra. These differences can be ascribed to perturbations of the physical properties of the protein upon adhesion to the surface of the silver colloids.

Using surface enhanced ROA (SEROA), novel results are achieved by combining Raman optical activity (ROA) and resonance surface enhanced Raman scattering (SERRS), applied on myoglobin. The novelty of this work is in reporting for the first time on chiral results of a study performed on a protein at single molecule level. This work, using silver nanoparticles and a laser excitation of 532 nm, only became feasible when the concentrations of nanoparticles, aggregation agent NaCl and the studied molecule were optimized in a series of systematic optimization steps. The spectral analysis has shown that the SERS effect behaves accordingly, depending on the concentration ratio of each component, i.e., myoglobin, Ag colloids and NaCl. Consequently, it is shown here that the SERS intensity has its maximum at a certain concentration of these components, whereas below or above this value the intensity decreases. The optimization results can be considered as a completion of the hitherto known phenomenon 'dilution effect', which only takes account of higher concentrations. Furthermore, the optimization of the parameters seems to be necessary for a successful SEROA measurement, which enables chiral study of a protein at the single molecule level, in which the concentration and acquisition time are no longer an impediment.