Tracking Conformational Changes in Phosvitin throughout a Crowding-Agent-Based Titration.

The sensitivity of Raman optical activity (ROA) towards small conformational changes is explored by tracking the structural changes in an intrinsically disordered protein-phosvitin-induced by different concentrations of crowding agent. It is shown that ROA is capable of tracking small conformational changes involving ß-sheet and α-helical secondary structural properties of the protein. Furthermore, it is indicated that the influences of the crowding agents employed, Ficoll 70 and dextran 70, on the structural properties of phosvitin differ significantly, with the structural changes induced by the presence of Ficoll 70 being more pronounced and already being visible at a lower concentration. The data also suggest that some spectral changes do not arise from a change in the secondary structure of the protein, but are related to differences in interaction between the phosphorylated residues of the protein and the sugar-based crowding agent.

Vibrational Circular Dichroism Sheds New Light on the Competitive Effects of Crowding and ß-Synuclein on the Fibrillation Process of α-Synuclein.

The effects of crowding, using the crowding agent Ficoll 70, and the presence of ß-synuclein on the fibrillation process of α-synuclein were studied by spectroscopic techniques, transmission electron microscopy, and thioflavin T assays. This combined approach, in which all techniques were applied to the same original sample, generated an unprecedented understanding of the effects of these modifying agents on the morphological properties of the fibrils. Separately, crowding gives rise to shorter mutually aligned fibrils, while ß-synuclein leads to branched, short fibrils. The combination of both effects leads to short, branched, mutually aligned fibrils. Moreover, it is shown that the nondestructive technique of vibrational circular dichroism is extremely sensitive to the length and the higher-order morphology of the fibrils.

Direct Measurements of the Crowding Effect in Proteins by Means of Raman Optical Activity.

The effect of crowding interactions on the structure of dephosphorylated α-casein was studied with Raman optical activity (ROA). It was found that ROA is sensitive to the structural changes in the protein, induced by the presence of crowding agents. This effect depends on the employed crowding agent and its concentration.