Addressing mechanism of fibrillization/aggregation and its prevention in presence of osmolytes: spectroscopic and calorimetric approach.

Understanding the mechanism of protein fibrillization/aggregation and its prevention is the basis of development of therapeutic strategies for amyloidosis. An attempt has been made to understand the nature of interactions of osmolytes L-proline, 4-hydroxy-L-proline, sarcosine and trimethylamine N-oxide with the different stages of fibrillization of hen egg-white lysozyme by using a combination of isothermal titration calorimetry, differential scanning calorimetry, fluorescence spectroscopy, and transmission electron microscopy. Based on thioflavin T fluorescence emission intensities and microscopic images, the nucleation, elongation, and saturation phases of fibrillization have been identified. Isothermal titration calorimetry and differential scanning calorimetry have enabled a quantitative analysis of the nature of interactions of these osmolytes with various conformational states of lysozyme at different stages of fibrillization/aggregation. It is concluded that interaction of the osmolytes with lysozyme fibrils at both the nucleation and elongation stages are important steps in the prevention of fibrillization/aggregation. Identification of the nature of interactions is a key step towards the discovery and synthesis of target oriented potential inhibitors of these associations. This study is a first report in which calorimetry has been used to address interaction of potential inihibitiors with the protein at different stages of fibrillization.