The fast paced, self-assembly of proteins in living organisms is essential for many biological processes. The formation of viral capsids, actin polymerisation in cells as well as DNA repair and maintenance all rely on intricate protein-protein interactions that result in self-assembly. While self-assembly is essential to life, when it goes wrong, it can cause serious problems. The unchecked self-assembly of proteins into amyloid fibres is a hallmark of several debilitating disease such as amyloidosis, Parkinson’s disease and Alzheimer’s.
However, the unnatural formation of amyloids is not always bad. Proteins that can quickly self-assemble into large, ordered structures with unique properties are of great interest for the production of novel biomaterials that are compatible with the human body and can be used for tissue engineering applications.
While understanding amyloid production offers tremendous value in several scientific disciplines, studying the details is difficult. Amyloids form through many protein-protein interactions and form intermediate structures as they grow. These interactions often occur too quickly for us to accurately measure and the intermediates don’t exist for very long. The key to understanding amyloid formation is to be able to analyse how the currently invisible intermediate steps occur and how they contribute to amyloid fibre formation.
A research team led by scientists at the University of Leeds investigated a technique for pushing the pause button on amyloid formation. In a recent publication they describe disulphide tethering whereby small molecules are used to covalently strengthen non-covalent interactions formed by amyloid intermediates. Once the amyloid intermediates known as oligomers form a complex with these small molecules, they become very stable and assembly is essentially halted.
The research team found that they were able to consistently stabilise large pools of oligomers that would ordinarily have been transient intermediates in the amyloid formation process. Once stabilised, the team were then able to carefully study the intermediates using the Microfocus Macromolecular Crystallography (MX) beamline, I24, at Diamond in conjunction with solution-state nuclear magnetic resonance (NMR) measurements. The results showed that the degree of stabilisation was dependent on the degree of tethering, implying that tethering the intermediates at different locations could give a slightly different picture of how the self-assembly process progressed. The research team were ultimately able to build up a far more detailed picture of the processes involved than had previously existed.
The study published in the Journal of the American Chemical Society highlights the power of covalent small molecules for studying specific protein-protein interactions. Previously, small molecules had been used extensively to interrogate the role of different proteins in biological processes and to aid in the structural studies of molecules that are difficult to form crystals. This study now adds a new way that small molecules can help to advance biochemical research.
This new method for studying the processes involved in diseases such as Parkinson’s and Alzheimer’s could help us find treatments. While primarily used as aids to study amyloid assembly, the small molecules studied here effectively halted amyloid production. This research shows that intermediate structures can be effectively frozen in time and analysed using X-ray crystallography, meaning powerful drug discovery approaches such as fragment screening can now be applied to amyloids.
The use of small molecules to trap transient states of amyloids for structural characterisation using X-ray crystallography is a new and exciting technique. It opens the door to visualising these processes and structure-based drug discovery.
Robin Owen, Principal Beamline scientist on I24.
Emma E. Cawood et al. Modulation of amyloidogenic protein self-assembly using tethered small molecules. Journal Of The American Chemical Society 142, 49. (November 2020). DOI: 10.1021/jacs.0c10629
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