Chemical and Biomolecular Engineering - Theses
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ItemProtein stability in shear flow( 2010)Given that globular proteins show a strong conformation-function relationship, the stability of a native protein structure is essential for its function. Aberrant proteins, resulting from structural instabilities in native protein conformations, and consequent aggregation, evolve a gain-of-function pathogenesis which has serious implications in industry and medicine. Therefore, it is important to appreciate the key factors that perturb the solution conformation of protein systems leading to aggregation. Considering the fact that proteins generally function in solution form, and those solutions have an inherent tendency to flow, knowledge of the stability of protein solutions in shear flow is essential. This thesis employs a combination of spectroscopic and microscopic techniques to study the conformational dynamics and morphological transformations of bulk peptide/polypeptide solutions in both uniform and heterogeneous velocity gradients. Preliminary studies in this thesis demonstrate that protein denaturation and subsequent aggregation can be probed using intrinsic protein fluorescence. The induction of protein aggregation was found to be greatly enhanced in heterogeneous flow regimes. Studies in a well defined flow field, Couette flow, revealed that the hydrodynamic stress generated in such flow regimes induce the unfolding of the helical segments of natively folded insulin; a prerequisite for aggregation and amyloid fibril formation. Further analysis of the shear-effect on α-helical conformations was performed using the homopolypeptide poly-L-lysine as a model protein system. The results reveal that the shear-induced unfolding of α-helical segments depends on both the shear rate and the duration of its application. An assessment of the chain-length-dependence of this phenomenon revealed that, contrary to classical theory, the strain in a given flow field varies inversely with the chain-length of α-helical poly-L-lysine. Collectively, the results provide new insight into existing theories in polymer physics. More importantly, it provides quantitative information on the conformational dynamics of peptide/polypeptide solutions in shear flow. This report is relevant to quality control measures during the commercial isolation and purification of protein products, and might help explain the role of shear stress, originating from pulsatile blood flow, in protein misfolding diseases and vascular disorders.