Thursday, November 21, 2024
11/21/2024
Abstract Protein self-assembly into structured fibrils plays both functional and dysfunctional roles in biology. We are investigating two classes of fibril forming proteins: collagen, a fibril that forms essential interactions with numerous proteins and extracellular matrix components for proper cellular function and α-synuclein (αS), an intrinsically disordered protein that self-assembles into oligomers and fibrils that are associated with debilitating synucleinopathies, such as Parkinson’s Disease. While in both cases, fibrils are often thought of as rigid, rather inert, entities, we have recently discovered conformational dynamics that profoundly impact their atomic- to-nano scale properties. Despite the importance of these fibrillar proteins, the molecular determinants of fibril- protein interactions and their impact in health and disease remain unanswered. Thus, the overarching objective of this proposal is to understand how molecular motions and surface properties modulate protein interactions at different assembly stages (monomer, oligomer, fibril), spatial extent (atomic to nanoscale), and temporal regime (picosecond to hours) to promote normal homeostasis or pathological disease states. The unifying theme of this proposal is that we are developing the key techniques and protocols necessary for fibril characterization that recognize the conformational plasticity and diverse interactions of these systems. We use a multifaceted approach integrating solution and solid-state nuclear magnetic resonance spectroscopy, atomic force microscopy, cryo-electron microscopy, computational methods, and link these to cellular experimentation. We are addressing the question of how collagen fibrils recognize their binding partners (we focus in particular on integrin, a key protein involved in platelet aggregation) despite the fact that many binding sites are hidden in the complex collagen fibril architecture. Beyond structure/function in healthy fibrils, we will investigate the impact of GlyX mutations in hereditary connective tissue disease such as Osteogenesis Imperfecta (brittle bone disease) and visualize for the first time how these defects impact on fibril assembly, structure and function. Although a very different biological system, we raise similar fibril interaction questions for αS: cell-to- cell propagation and templated seeding of endogenous αS monomers by fibrils increases the number of fibrils and is one of the primary factors in disease progression. This interaction mechanism is not understood and we will investigate this by first characterizing amyloid surfaces from the atomic to the nano-scale and then visualizing the interactions of the monomers with them. Results will shed light on the nature and specificity of collagen and αS interactions, the biophysical and biological impact of disease-affiliated collagen mutations, and mechanisms of pathological cell-to-cell propagation and seeding of αS aggregates. Elucidating novel interaction mechanisms will aid in design of new therapeutic strategies to rescue compromised collagen interactions or pathological αS aggregation in devastating neurodegenerative disease.
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