Mechanistic Studies of Amyloid Propagation Auto-catalytic functions of aggregation

Proteins are perhaps the most diverse group of molecules currently known. These hetero-polymers consist of the twenty canonical amino acids. Proteins cover an unfathomable large sequence space and the structural diversity of proteins is equally impressive. Despite this extreme diversity, a seemingly intrinsic feature of these molecules is the ability to aggregate into well-ordered fibrillary species called amyloid fibrils. Formation of such fibrils in human cells is associated with onset of amyloid diseases, among these are neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s Disease (AD). Structural characterization of such fibrils has advanced considerably, however thorough understanding of the microscopic steps associated with aggregation remains elusive.

This thesis mainly focuses on describing aggregation mechanisms in the perspective of structural understanding of the amyloid state, as to infer molecular level interpretation of the aggregation reactions. To this end surface based biosensing, in the form of Quartz-Crystal Microbalance with Dissipation (QCM-D), has played a crucial role in accurate measurements of isolated aggregation mechanisms, which is otherwise extremely challenging using current bulk methodologies common in the field.

Firstly, I address the necessity for easy and quantitative characterization of amyloid structural ensembles. Atomic Force Microscopy (AFM) is a scanning probe method, which demonstrates high signal-to-noise ratio for current commercial instruments. AFM images of surface deposited fibrils allow for single-fibril characterization enabling study of population heterogeneity. I present a tool developed for analyzing such images in concert with open-source AFM analysis software. Using the tool, fibrils are characterized by their length, filament height and helical twist length, useful for both seeded kinetic studies and equilibrium studies.

Secondly, we apply the afore-mentioned QCM-D platform to investigate the role of dynamic regions of amyloid fibrils in growth mechanisms. Here we demonstrate that upon proteolytic removal of these dynamic regions, secondary growth processes can be enabled. We utilize the full suite of information provided by the QCM-D and perform a novel time-resolved analysis of the observed growth-mechanics providing detailed insights into the molecular information otherwise hidden in our measurements.

Thirdly, we apply a protein engineering approach from classical protein folding studies, Φ-value analysis, to the amyloid elongation reaction. These studies characterize thermodynamic stability and kinetics of an amyloid model system and a cohort of mutants at an unprecedented level. This approach allows us to characterize the transition state (TS) of the amyloid elongation reaction and construct an energy landscape model. This allows us to encompass the currently most prominent theoretical and experimental studies of amyloid elongation into a single model.

Finally, I construct and demonstrate an experimental pipeline for studying liquid assembly and subsequent solidification of fibril forming Antimicrobial Peptides (ffAMPs) and single stranded DNA. This pipeline utilizes recent advances in methods for study of Liquid-Liquid Phase Separation (LLPS) from our laboratory to create a high-throughput characterization platform. While currently in a preliminary state, this platform will assist in identifying correlations between condensation, solidification and fibril structure formation of these short peptides.

This work establishes new insight into the auto-catalytic functions of amyloid assemblies and the approaches outlined will hopefully assist in pushing the scientific community towards a molecular understanding of these mechanisms.