Investigating the physicochemical properties of coacervates formed by intrinsically disordered proteins.

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In the last 10 years there has been an explosion of research into liquid condensates formed by intrinsically disordered proteins. These protein condensates, or droplets, often display biochemical activity such as RNA sequestration. Ddx4 is an RNA processing protein with an intrinsically disordered domain that, at high enough protein concentrations, will form protein rich droplets in aqueous solution. These droplets can act as biomolecular filters in vitro, preferentially absorbing shorter regulatory RNA's and melting short DNA duplexes. The mechanism of this biochemical activity is currently unknown.There has been some work relating protein sequence to the formation and activity of liquid droplets, but this is still in its infancy. There is also little to no characterisation of droplet interfaces, yet their structure determines their permeability to cofactors, wetting behaviour and rate of droplet growth. This project will build on previous research on Ddx4, Dr TJ Nott has three high impact papers published on this protein. Ddx4 reliably forms droplets that do not show appreciable ageing phenomena, unlike other condensate forming proteins such as LAF1 and FUS. Bespoke protein design is an overarching goal of synthetic biology. By uncovering the mechanisms that govern the behaviour of Ddx4 I will enable to rational design of a condensate forming protein with tuneable properties. Research Objectives1. Selective interactions of Ddx4 droplets with oligonucleotidesIt is known that Ddx4 droplets will absorb oligonucleotides in a manner that is selective for their secondary structure. Conformational changes of oligonucleotides upon absorption will be investigated. I will record changes in the Forster resonance energy transfer (FRET) signal for oligonucleotides labelled at the 5' and 3' positions with fluorophores upon absorption into protein droplets. 2. Interfacial properties of protein dropletsIn collaboration with Prof Dirk Aarts, methods from colloid science will be used to study the interfacial properties of ddx4 droplets. The goal is to find methods and models from the field of soft condensed matter that yield tangible, biologically relevant conclusions about droplet interfaces. The mechanism of droplet nucleation will be studied by investigating the spatial distribution of droplets shortly after their formation, exploring this over a range of parameters. An assay for surface properties will be developed by studying the nucleation and wetting of droplets on a range of functionalised surfaces. I hope to use phase contrast microscopy to image droplets that lack any fluorescent tag. 3. Relating droplet functionality to sequenceThe sequence features that effect protein-DNA interactions and interfacial properties of Ddx4 droplets will be determined. Through cloning and site directed mutagenesis, I will generate a range of mutant Ddx4's. The modifications of interest are as follows. - Point mutations of residues known to contribute to the condensation.- Elongation of the Ddx4 protein.These modified Ddx4's will then be subjected to the same methods as in points 1 and 2. I will investigate the degree to which the selective oligonucleotide absorption and interfacial properties of Ddx4 are coupled, and therefore whether these two properties are independently tuneable. I hope to introduce a droplet forming protein with the ability to encode selectivity in its in vitro interactions with olignucleotides. Because these droplets often display biochemical activity, such as RNA sequestration, they will have applications in the design of synthetic tissues for biomedical applications. The DPhil project falls within the EPSRC 'Biophysics and Soft Matter Physics' and 'Synthetic Biology' research areas. Accompanying the research themes, I will also spend 2 months on an industrial placement at OxSyBio who are developing both a 3D cell printing platform and are studying synthetic tissues formed from active droplets.