Assembly mechanisms of type I collagen aggregates

grantor: University of TorontoThe solution-phase association of biological molecules to form aggregates has been studied from a variety of perspectives, such as the flocculation of polymers, association of colloids and the aggregation of proteins. The latter subject has been the target of particular attention, mainly because of its importance in biomedical applications as well as its implication in diseases such as spongiform encephalopathies and Alzheimer's disease. Investigating protein aggregation at a fundamental level, however, is often complicated by the poorly defined structures of the aggregation products formed, as well as by the difficulty in working with biological systems. In this thesis, studies of Type-I collagen, a rod-like protein which can undergo 'in vitro' aggregation into highly-ordered protein structures, have been carried out with a view towards determining the mechanisms by which protein aggregates form. Two particular aggregate structures have been investigated. The first, a block-like aggregate called segmental long spacing collagen, was formed by the addition of nucleotide triphosphates to collagen monomers. By analyzing aggregate structures, formation kinetics and growth thermodynamics, it was concluded that these aggregates form via a hierarchical growth mechanism involving the formation of a stable intermediate and subsequent fusion of these intermediates. The second form of aggregate investigated was a fibril referred to as fibrous long spacing collagen. In this case, particular insight was made into elucidating the structure of these fibrils using the atomic force microscope. Results obtained again suggest that this aggregate forms via a hierarchical mechanism, first forming stable 'protofibrils', which then merge in a complex manner to produce the final structure. In the final Part of this thesis, the capabilities of the atomic force microscope in performing imaging of dynamic, biological processes in real time was examined, and was used to image the enzymatic digestion of collagen fibrils by the protein collagenase. The utility of the atomic force microscopy for 'in situ' investigation of complex processes such as collagen formation is discussed with a view towards applying this technique to future work.Ph.D