Intercations between Polypeptide and Protein molecules
Date Issued
2010
Date
2010
Author(s)
Lai, Jun-Kun
Abstract
Proteins are essential elements for living organisms and play a crucial role in various physiological activities. An enzyme is a protein molecule that serves as a biological catalyst. Proteins/enzymes with correct conformations are able to serve appropriate biological functions. Proteins that misfold may not only lose their normal biological function but also form aggregates which lead to a variety of diseases.
In this thesis, we attempt to explore the interactions between the polypeptides and protein molecules. The preservation of protein conformation was carried out by copolypeptides with different compositions through various processes such as entrapment, or adsorption. Moreover, we examined the effects of polypeptides on folding, structural changes, aggregation, and amyloid fibrillation of proteins.
In the first part of the thesis, we report the immobilization of a model enzyme, papain, within silica matrices by combining vesiclization of poly-L-lysine-b-polyglycine block copolypeptides with following silica mineralization. The polypeptide mediated silica-immobilized enzyme exhibits enhanced pH and thermal stability and reusability, comparing with the free enzyme and the vesicle encapsulated enzyme (e.g. after 48 hr incubation at 25°C, the percentage residual activities for the immobilized and the untreated papain samples were found to be ~68.5% and ~29.6% of that of the free papain at 0 hr, respectively). The enhanced enzymatic activity in the immobilized enzyme is due to the confinement of the enzyme in the polypeptide mediated silica matrices. Kinetic analysis shows that the enzyme functionality is determined by the structure and property of silica/polypeptide matrices.
In the second part of the thesis, with hen egg-white lysozyme and bovine insulin as the model systems, we show the results regarding the influences of two random copolypeptide D,L-lysine-co-glycine and D,L-lysine-co-L-phenylalanine on the in vitro protein fibrillation. Our TEM and ThT fluorescence results show that the observed inhibitory effects on amyloid fibrillation are significantly dependent on the amount and the composition ratio of polypeptide chains. For instance, the percentage reduction in hen lysozyme fibrillation was found to be approximately 35 % or 65% for the case of 1 mM or 2 mM random copolypeptide, respectively. The addition of 0.5 mM or 1 mM random copolypeptide results in approximately 25 % or 80% reduction, respectively, in fibrillogenesis derived from bovine insulin. The copolypeptides with a higher fraction of glycine or L-phenylalanine residue exhibit higher inhibitory potency against fibril formation. Moreover, we examine the structural changes in both proteins and inhibition mechanisms through CD spectroscopy, ANS fluorescence, intrinsic fluorescence spectroscopy, and SDS-PAGE. The major driving forces for the association of HEWL and copolypeptides are likely hydrogen bonding and hydrophobic interactions. We believe that the outcome of this work may contribute to the understanding of molecular mechanism(s) of the fibril formation and provide potential treatment strategies against the amyloid formation associated with amyloid disease.
Subjects
lysozyme
amyloid fibril
insulin
papain
copolypeptide
inhibition
vesicle
Type
thesis
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