Understanding glycoprotein behaviours using Raman and Raman optical activity spectroscopies: Characterising the entanglement induced conformational changes in oligosaccharide chains of mucin

We illustrate the great potential of Raman and ROA spectroscopies for investigating the structure and organisation of glycoproteins and the complex matrices they can form. In combination these spectroscopic techniques are sensitive to changes in conformation revealing details of secondary and tertiary structures, probing hydrogen bonding interactions, as well as resolving side chain orientation and the absolute configuration of chiral substructures. To demonstrate this potential we have characterised the structural changes in a complex glycoprotein, mucin. Spectral changes were observed during the entanglement transition as the mucin concentration was increased. By applying two-dimensional correlation analysis (2DCos) to the ROA and Raman concentration-dependent spectral sets delicate transitions in mucin conformation could also be determined. From ∼ 20-40 mg/ml conformational transitions assigned mainly to the sugar N-acetyl-d-galactosamine (GalNAc), which is the linking saccharide unit to the protein backbone, were monitored. Further changes in local oligosaccharide conformation above 40 mg/ml were also monitored, together with other structural transitions observed in the protein core, particularly β-structure formation. Consequently, these spectral techniques were shown to monitor the formation of transient entanglements formed by brush-brush interactions between oligosaccharide combs of mucin molecules identifying changes in both carbohydrate and protein moieties. This work clearly shows how these methods can be used to elucidate fresh insights into the complex behaviour of these large complex molecules

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