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Determination of molecular dynamics in membrane proteins and protein fibrils using novel solid-state NMR methods

Final Report Summary - DYNAMIC PROTEINS (Determination of molecular dynamics in membrane proteins and protein fibrils using novel solid-state NMR methods)

Many of age-related neurodegenerative disorders such as Alzheimer's disease (AD), parkinson's disease (PD), type II diabetes mellitus are characterised by accumulation of insoluble proteinaceous deposits. These aggregates are composed of beta-sheet rich fibrillar aggregates, known as amyloid fibrils. In parkinson's disease, alpha-Synuclein (AS) fibrils are the main constituent of the brain of patients. AS is a monomeric protein that forms fibril in a nucleation-dependent manner. It is imperative to elucidate structure-folding-dynamics relationship to understand the role of various species of AS in aetiology of Parkinson's disease.

As amyloid fibrils are intrinsically non-crystalline and water-insoluble, therefore, we applied two-dimensional solid-state NMR to elucidate the atomic-level structural organisation of straight-type alpha-fibrils. We used various set of C-C and N-C correlations to unambiguously assign resonances coming from the core of the fibril (38-103), afterward; secondary structural elements were calculated using chemical shifts. Further, we probed the protofilament arrangement on a residue-specific level in reference to an aqueous environment. The data obtained from these approaches enabled us to deduce arrangement for beta-strands and we show that the core region of AS is devoid of highly mobile bulk water.

Subsequently, supra-molecular arrangement of the beta-strands within the beta-sheets was investigated by NNHC experiment performed on a mixed labelled sample to deduce the alignment of beta-strands within the fibril beta-sheets at individual residues-level. We demonstrated that the beta-sheets are arranged in parallel and in-register fashion.

Finally, some inter-strand distances were measured to propose a three-stranded superpleated arrangement for AS fibrils.

Increasing evidences suggest that the prefibrillar intermediates are the primary causative agents in neurodegeneration. Despite their criticality in curing neurodegeneration, little is known about the structural and motional aspects of such intermediates. This is because of their transient nature and aggregation prone behaviours. We prepared on-pathway oligomers of alpha-synuclein with higher concentrations and characterised their structural and functional properties by a combination various biophysical techniques such as, ThT fluorescence, EM, AEM, electrophysiological measurements, and solid-state NMR. Further, we were able to demonstrate that these AS oligomers form ion channels with well-defined conductance states in a variety of artificially constituted membranes, and have nonfibrillar beta-structural preferences.

Next, we investigated the effect on designed small molecules on the kinetics and morphology of AS fibril formation. The designed molecules, diphenylpyrazole (DPP) and its derivatives, can dissolve AS fibrils and on-pathway oligomers, whereas, it appears to stabilising off-pathway oligomers.

In view to gain a detailed mechanism of action of DPP, we prepared a frozen solution of off-pathway oligomers with DPP and investigated their structure using solid-state NMR. We recorded a set of one- and two-dimensional spectra of off-pathway oligomers and compared them with monomers and fibrils spectra. Off-pathway oligomers generated a distinctly different spectrum with less beta-type characteristics than fibrils but were more ordered than the on-pathway oligomers. Two-dimensional spectra provided an insight into structural organisation of these species.

During the course of this project, we targeted AS, a protein involved in second most common neurodegenerative disease, the Parkinson's disease. Our results led towards a better understanding for the structural and motional details of fibril organisation and structural preferences of on-and off-pathway oligomers.

These findings might lead to a better understanding for the structural biology of various states of AS and will be directly applicable to mankind in terms of better drug development.