Mechanistic insights on protein aggregation and phase separation

Thesis

Proteins are essential structural and functional components of cells and act at a range of assembly states, from single proteins to macromolecular complexes. The pathological aggregation of proteins into amyloid fibrils and other morphologies of aggregates is a hallmark of several human diseases, many of which are neurodegenerative disorders like Alzheimer’s and Parkinson’s and pose a global health burden due the limited therapeutic approaches.

Proteins carry out their function not only in very well defined structured states, which is the basis of the structure-function dogma, but also by exploiting the unique properties of conformationally heterogeneous disordered states. In particular, the recently discovered phenomenon of protein phase separation, in which proteins and nucleic acids form a distinct phase (a membraneless organelle or biomolecular condensate) from the surrounding bulk environment, is emerging as a key compartmentalisation mechanism in cells, alternative to membrane-bound compartments, with a wide range of functions and unique properties.

In this thesis, I present the results of applying biophysical approaches, mainly nuclear magnetic resonance (NMR) and modelling, to the processes of protein aggregation and phase separation, with the aim to gain a better understanding of those at the molecular level. Chapter 1 introduces the topics of study, covering the fundamental aspects as well as the specific systems of study in more detail.

In chapters 2 through 4, I present the application of a recently developed NMR- based chemical kinetics method to the mechanism of aggregation of the protein tau and its inhibition by a peptide. In chapter 2, I present the R3 model system of tau as a biologically relevant model for tau which is amenable to our method. Chapter 3 is concerned with the mathematical modelling challenges and presents further developments to the initial method. Finally, in chapter 4, I present the mechanisms of aggregation and inhibition obtained by applying this modelling strategy to R3 and the peptide D-TLKIVW. The primary aggregation of tau R3 includes dimerisation and the formation of a tetrameric nucleus that elongates into fibrils, which can fragment. We find that D-TLKIVW, originally developed as an elongation blocker, has a more nuanced mode of action than expected and targets in particular the nucleus.

In chapter 5, I focus on the phenomenon of phase separation and in particular the hypothesis that condensates constitute a distinct chemical environment, a solvent. We investigate this by studying the partitioning of small molecules into condensates of two phase separating proteins, DDX4 and FUS. The results include the development of several NMR-based methods to measure partitioning, as well as reveal chemical trends in the relative partitioning of a set of nucleotides.

Authors: Casablancas Antràs, V

• DOI: 10.5287/ora-qq77bd2gy
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: protein aggregation; phase separation; tau; NMR; mechanism of action
• Subjects: Protein-protein interactions; Physical and theoretical chemistry; Biochemistry; Chemical kinetics; Reaction mechanisms (chemistry); Drug interactions