Investigating the physicochemical properties of coacervates formed by intrinsically disordered proteins.
Lead Research Organisation:
University of Oxford
Department Name: Synthetic Biology DTC
Abstract
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 Objectives
1. Selective interactions of Ddx4 droplets with oligonucleotides
It 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 droplets
In 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 sequence
The 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.
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 Objectives
1. Selective interactions of Ddx4 droplets with oligonucleotides
It 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 droplets
In 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 sequence
The 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.
Planned Impact
The emerging and dynamic field of Synthetic Biology has the potential to provide solutions to some of the key challenges faced by society, ranging across the healthcare, energy, food and environmental sectors. The UK government has recently a "Synthetic Biology Roadmap", which presents a vision and direction for Synthetic Biology in the UK. The report projects that the global Synthetic Biology market will grow from $1.6bn in 2011 to $10.8bn by 2016. It highlights that there is an urgent need for the UK to develop the interdisciplinary skills required to take advantage of the opportunities provided by Synthetic Biology.
The challenge to the academic and industrial research communities is to develop new translational approaches to ensure that these potential benefits are realised. These new approaches will range across the design and engineering of biologically based parts, devices and systems as well as the re-design of existing, natural biological systems across all scales from molecules to organisms. The techniques will encompass not only individual cells, but also self-assembled biomimetic systems, engineered microbial communities and multicellular organisms, combining multiple perspectives drawn from the engineering, life and physical sciences.
Realising these goals will require a new generation of skilled interdisciplinary scientists, and the training of these scientists is the primary goal of the SBCDT. Our programme will give the breadth of coverage to produce a "skilled, energized and well-funded UK-wide synthetic biology community", who will have "the opportunity to revolutionise major industries in bio-energy and bio-technology in the UK" (David Willetts, Minister for Universities and Science) in their future careers. This will be made possible through genuine inter-institutional collaboration in partnership with key industrial, academic and public facing institutions.
The potential impact of the SBCDT, and its potential national importance, are very therefore high, and the potential benefits to society are significant.
The challenge to the academic and industrial research communities is to develop new translational approaches to ensure that these potential benefits are realised. These new approaches will range across the design and engineering of biologically based parts, devices and systems as well as the re-design of existing, natural biological systems across all scales from molecules to organisms. The techniques will encompass not only individual cells, but also self-assembled biomimetic systems, engineered microbial communities and multicellular organisms, combining multiple perspectives drawn from the engineering, life and physical sciences.
Realising these goals will require a new generation of skilled interdisciplinary scientists, and the training of these scientists is the primary goal of the SBCDT. Our programme will give the breadth of coverage to produce a "skilled, energized and well-funded UK-wide synthetic biology community", who will have "the opportunity to revolutionise major industries in bio-energy and bio-technology in the UK" (David Willetts, Minister for Universities and Science) in their future careers. This will be made possible through genuine inter-institutional collaboration in partnership with key industrial, academic and public facing institutions.
The potential impact of the SBCDT, and its potential national importance, are very therefore high, and the potential benefits to society are significant.
