Looking at protein dynamics and thermodynamics through the lens of exact NOE and The pH-sensitive nature of the structural polymorphs of α-synuclein fibrils revealed by cryo-EM
Embargoed until 2026-03-03
Author
Date
2023Type
- Doctoral Thesis
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Abstract
Nuclear Magnetic Resonance (NMR) spectroscopy and Cryogenic Electron Microscopy (Cryo-EM) are two of the most widely used techniques for determining the three-dimensional structure of macromolecules. On the one hand, NMR spectroscopy is the most effective method for investigating the structural and dynamical information of small to medium-sized (< 50 kDa) proteins. On the other hand, cryo-EM is particularly adept at acquiring structural information of large protein complexes at near-atomic resolution (< 3 Å) in their native state.
This thesis has been split into two parts based on the structural biology technique utilized and the class of proteins being investigated. Part I of this thesis examines the potential of exact NOE (eNOE), an NMR-based method developed in the Riek Lab, to enable multi-state structural studies and shed light on the conformational dynamics and thermodynamics of proteins. In chapter 1, we provide a brief overview of the principles of NMR spectroscopy and the methodology behind the eNOE. Unlike traditional NMR-based structure determination methods, eNOEs can provide distance restraints for structure calculation with an unprecedented accuracy of up to 0.1 Å. These distances are subsequently used to refine the structures and potentially detect the presence of multiple conformations of a protein/protein complex. eNOE method development is ongoing, and many applications have been added to the eNOE toolbox over the years, a few of which are discussed in Chapters 2, 4 and 5 of this thesis.
eNOEs can also be used to determine precise intermolecular distances, enabling the study of protein-ligand complexes. In chapter 2, we present the eNOE-determined two-state structure of the HDM2-Nutlin 3a complex. HDM2 is a highly versatile protein and plays a vital role in cancer research. It changes its conformations and dynamics to adapt its function, especially in the vicinity of its large binding site (~ 1000 Ų) which only fully reveals itself in the presence of a ligand. Nevertheless, the underlying principles that govern these polymorphic capabilities remain poorly understood. One potential inhibitor of HDM2 is Nutlin 3a and deciphering the structure of this complex can help further drug discovery.
In chapter 3, we provide a description of an established approach to investigate conformational dynamics at (ps-ns) timescale, using NMR-based relaxation experiments. Utilizing this approach in conjunction with eNOE multi-state structural calculation can provide secondary evidence for the existence of multiple protein states and their role in allosteric modulation in the presence of ligands. This has been demonstrated in the study on the allosteric mechanisms of the PDZ domain described in this chapter.
Nuclear Magnetic Resonance Molecular Replacement (NMR²), developed by Orts et al., is a hybrid approach to determine the structure of protein-ligand complexes, utilizing a previously determined structure of the target protein and combining it with the spatial information extracted by intermolecular eNOEs to identify the binding pocket of the protein and the orientation of the ligand inside it. In chapter 4, we present an alternative, time-efficient approach to extract quantitative restraints for NMR² structure calculation. This approach can match the predictions of conventional, eNOE-based NMR² in the case of strong binders and possibly surpass them in the case of weak binders with few distance restraints.
Due to the exact and quantitative nature of the eNOE cross-relaxation rates, eNOE can also be used as a probe to access information about a protein’s energy landscape and equilibrium populations at a local, residue level. This can potentially provide us with unprecedented insight into mechanisms as diverse as allosteric signaling, enzymatic catalysis and drug binding. In chapter 5, we present a proof-of-concept study on a model protein, PIN1-WW domain, with an already existing two-state eNOE structure. The evolution of the eNOE rates was modeled with increasing temperature under a two-state thermodynamics paradigm to extract thermodynamic parameters like the enthalpy difference, ΔH, and the entropy difference, ΔS, between the two states.
In Part II of this thesis, we switch to cryo-EM to elucidate the structure of protein assemblies called amyloid fibrils. They are a unique class of self-assembled protein/peptide filamentous aggregates containing cross β-sheet motifs that have been linked to several dozen human diseases. Some of the most studied amyloid-related diseases are synucleinopathies, which include conditions such as Parkinson’s disease (PD), multiple systems atrophy (MSA), and dementia with Lewy bodies (DLB). The accumulation of α-synuclein amyloid fibrils in high concentration within inclusion bodies, such as Lewy bodies (LB), is a defining characteristic of synucleinopathies. Chapter 6 introduces the workflow of cryo-EM structure determination, with an emphasis on the helical reconstruction method used to determine the structure of amyloid fibrils of α-synuclein.
In contrast to folded proteins, the structural/energetic landscape of neurodegenerative disease-associated amyloids, including α-synuclein, is highly diverse and gives rise to structural polymorphs. In Chapter 7, we include a study that explores the effect of changing pH and salt concentration on the α-synuclein fibril formation. The atomic resolution cryo-EM structures of α-synuclein fibrils determined under these conditions demonstrate that the buffer environment strongly participates in polymorph formation and selection. Lowering the pH results in the formation of a distinct class of α synuclein polymorphs, while increasing the salt concentration influences protofilament assembly and the population of the polymorphs. These findings, alongside that of Thioflavin T (Thio-T) aggregation kinetics and limited proteolysis-coupled mass spectrometry (LiP-MS), suggest that secondary nucleation is the primary driver of α-synuclein fibril formation. Show more
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https://doi.org/10.3929/ethz-b-000601341Publication status
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ETH ZurichSubject
NMR spectroscopy; Cryo-electron microscopy; Protein Dynamic; Exact NOE; Alpha SynucleinOrganisational unit
03782 - Riek, Roland / Riek, Roland
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