Spectroscopic Studies of Proteins and Their Interactions with Lipids and Small Molecules
Year: 2014
Type: dissertation
Abstract: The amyloid-? (A?) peptide, a major component of the insoluble plaques associated with Alzheimer�s disease (AD), is the cleavage product of the transmembrane (TM) amyloid precursor protein (APP) and is known to possess an amiphiphilic nature. The full length A? is intrinsically disordered under physiological conditions, but could aggregate over time to form soluble neurotoxic oligomers and eventually insoluble fibrils. Although great research interest has been drawn to the underlying mechanism of the development of AD, controversy remains and there is still a lack of established medical treatment. Previous studies have shown that A?(25-40), the hydrophobic and TM fragment of A?, behaves similarly to the full length A?. The naturally occurring polyphenolic compound, myricetin, has been proven to inhibit the aggregation progress of A?(25-40) in vivo. To better understand the aggregation propensity of A? itself and the interaction mechanism between A? and myricetin, two site mutants, A?(25-40) G37A and I32A, have been designed to interrupt the peptide backbone hydrogen bonding as well as reduce hydrophobic interactions. Further thioflavin T (ThT) assays and circular dichroism (CD) results indicate that the hydrophobic interactions may be crucial in A?(25-40)�s intrinsic aggregation propensity and also play an important role in the interaction between myricetin and A?. Deep ultraviolet resonance Raman (dUVRR) and size exclusion chromatography (SEC) experiments suggest that the various initial oligomeric states may contribute to the altered A?(25-40) property. Derived from the membrane anchoring C-terminus of APP, A? is known to spontaneously associate with different types of lipid bilayers. To better understand the role of peptide-membrane interactions in AD, knowledge about how membrane characteristics affect binding, solvation, and secondary structure of A? is in demand. Employing a combination of CD and dUVRR spectroscopies, A?(25-40) was found to spontaneously associate with anionic lipid bilayers and the structure of the membrane associated A?(25-40) was dependant on both the hydrophobic thickness of the bilayer and duration of incubation. Further studies regarding the full length peptide indicate similarity between A?(25-40) and A?(1-40). After spontaneously associating with the negatively charged lipid bilayer, the full length A?(1-40) initially adopts a mixed conformation of ?-helical and disordered structures. The mixed structures then convert to more ?-sheet structure over longer time-frames, compared to the initial stage. ?-Sheet structure appears prior to the unwinding of the ?-helix, implying a process in which ?-sheet structure, formed initially from disordered regions, destabilizes membrane solvated helical structure and then leads to the escape of peptide from the membrane surfaces. The generation of A? involves a ?-secretase cleavage process within the membrane environment. However, there is no thorough understanding of how ?-secretase locates A? and what structural changes are associated with this event. In fact, there is very limited knowledge about the structure of TM proteins, compared to their well studied soluble counterparts. Although TM proteins not only participate in disease development but also perform fundamental biological functions in human physiology, the application of traditional high resolution structural analysis strategies to TM proteins is quite limited due to their relatively large sizes, complex structural conformation, and the surrounding native lipid environment. Vibrational spectroscopy methodologies, like dUVRR spectroscopy, offer an alternative approach to reveal the lipid solvated protein secondary structures as well as to monitor TM protein dynamics. Previous studies have produced a series of TM helical peptides to monitor the association and structure conformatioal equilibration between the membrane and peptides. In order to better define dUVRR characteristics of membrane associated/solvated protein secondary structure and establish potential quantitative relationships between the spectral response and the size of the protein TM domain, a series of model peptides, poly-(LA)x, were designed based on known TM helical peptides. It is found that both the length of the TM region of the peptide and the hydrophobic thicknesses determined by the lipid bilayer play important roles in the conformation of the final adopted structure, while the overall peptide secondary structures are predictable based on the TM region/hydrophobic thicknesses ratio and lipid bilayer composition.
Author: Jian Xiong
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2566552886