The Mechanism Of Amyloid Protein Fibrillation And Relevant Molecular Interactions

Amyloid protein fibrillation is a unique type of protein self-assembling process through which protein molecules form fibrillar aggregates with the characteristic cross-?structure.The basic structural unit of protein fibril contains a pair of?-sheets aligned parallel to each other.The width of such fibril is in nanometer scale and its length can be tens of nanometers or be in micron scale.Therefore,it can be considered as a protein nanowire.In history,these protein fibrils had been considered to be made of starch by mistake.This is why we can call them amyloid fibril.Amyloid protein fibrillation has been a hot topic in the field of protein sicence in the past two decades.This is primarily due to the fact that this phenomenon is related to about 40human diseases,including some well-known disorders such as Alzheimer’s disease,Parkinson’s disease,type II diabetes.There are currently no cure for these diseases.In addition,amyloid protein fibrils possess some excellent materials properties.In recent years,people have developed some novel nanomaterials based on such fibrils.These materials have shown great promise in a variety of fields such as sensing,catalysis,cell culture,retroviral transduction,and drug delivery.In this thesis,we ain to study the mechanism of amyloid protein fibrillation and relevant molecularinteractionsbyvariousanalyticalmethodssuchas infrared probe technique,fluorescence spectroscopy and so on.Namely,we studied the effect of Hofmeister ions on amyloid protein fibrillation,the kinetic mechanism of the binding of fluorescence dye onto amyloid protein fibrils,and the interaction between model amyloid peptide and graphene oxide.The details of our work are summarized below.1.Using the amyloid fibrillation system by hydrolzed lysozyme peptide fragments as the model system,we for the first time studies the impacts of a total of nine Hofmeister cations(namely,NH₄⁺>K⁺>Na⁺>Cs⁺>Li⁺>Rb⁺>Mg²⁺>Ca²⁺>Ba²⁺)on amyloid protein fibrillation.Through kinetic analysis on the fibril growth curve to obtain kinetic parameters,we analyzed the relationship between the kinetics of amyloid protein fibrillation and the order of Hofmeister serises.In addition,we also investigated the effects of Hofmeister series ions on the yield of fibrillation and the morphology of fibril.We conclude that there is no clear correlation between amyloid protein fibrillation and the order of Hofmeister serises.Yet,the Hofmeister cations show some impacts on on the yield of fibrillation and the morphology of fibril.2.Using stopped-flow kinetic apparatus,we studies the kinetic binding mechanism of ThT fluorescence dye onto lysozyme amyloid fibril.Through newly designed kinectic approach,by keeping lysozyem fibril in excess and monitoring the decay of ThT with UV-Vis spectroscopy,we have deduced the kinetic equations of ThT binding onto lysozyme fibril and proposedaone-stepparallelbindingkineticmechanism.Furthermore,in fluorescence-detected stopped-flow kinetic study,we showed that there were two types of ThT binding sites on the lysozyme fibril.3.We chose a seven-residue short peptide（referred to as GA）as the model for?-amyliod peptide and studied the adsorption of GA onto graphene oxide and the interaction mechanism.GA induced quick aggregation of graphene oxide,the surface of graphene oxdie becomes rough,and the zeta potential of graphene oxide also changed significantly.Using infrared probe technique combined with temperature dependent infrared study,we invevestigated the spectroscopic changes of CN infrared probe before and after GA adsorption.We found that CN and water formedπ-hydrogen bond on the surface of graphene oxide.Combined with Raman spectroscopic evidence,we proposed that GA adsorbed on graphene oxide surface as a monolayer throuhg chemsorption and the aromatic group of GA molecule formed off-center face-to-faceπ-stacking structure with graphene oxide.