Better together: constructing tuneable fluorescent protein sensors for cellular imaging of protein-protein interactions.

About the Project

Protein-protein interactions (PPIs) commonly occur in biology with ~650,000 contributing to processes critical to human life. From cancer to dementia, their dysregulation contributes to many disease states. PPIs occur when one protein interacts with another resulting in functional changes that have repercussions on the cell. Their importance means PPIs are studied across the different bioscience fields and many tools developed to investigate them.

Fluorescence-based methods allow researchers to study these innately dynamic processes in a proteins’ natural environment, the cell, in real time leading to many new insights. Genetically-encoded probes called fluorescent proteins have proved pivotal to investigating PPIs in the cell but the underlying methods are currently limited to monitoring interactions between two different proteins; self-association of the same proteins cannot be easily measured. This is major issue as many PPIs, arguably the majority, involve the self-association of identical proteins. Thus, researchers cannot investigate this vital piece of the PPI story within a protein’s natural cellular context.

The aim of your project will be to design and engineer a new set of fluorescent proteins that change their function on self-association (homo-dimerisation) and to use these new tools to monitor formation of homo-oligomer complexes. The Jones lab has recently shown that designed self-association of green fluorescent proteins can lead to the “switching on” of fluorescence output [References 1-3]. You will use existing structural knowledge together with computational protein design and directed evolution to generate a new protein-protein interface to promote GFP self-association.

You will initially use a model small molecule-dependent dimerisation system to assess the properties your new fluorescent proteins, and use biophysical analysis and structural biology to understand the molecular basis of action. Those GFP variants with the required properties will then be applied in mammalian cells to monitor protein-protein interactions using state-of-the-art fluorescence microscopy (in situ cell imaging).

You will then apply your new tool to monitor oligomerisation events the biologically important NF-kappaB complex that controls transcription and is associated with a variety of different disease states such as cancer, inflammation and autoimmune diseases. You will combine your new dimerisation-dependent GFP tagged NF-kappaB core components with other fluorescent protein tagged proteins to monitor in real time events associated with NF-kappaB assembly using state-of-the art fluorescence microscopy.

By the end of the project, you will have developed tool for broader use in the life sciences and applied it to understand an important biological complex.

Techniques: computational analysis, protein engineering (including design and molecular biology), protein chemistry (purification and analysis), protein 3D structure determination, biophysical analysis (various spectroscopy methods, including single molecule analysis), cell biology (including mammalian cell culture), fluorescence microscopy.

Contact details for supervisor- Prof Dafydd Jones

Email – jonesdd@cardiff.ac.uk

Phone Number - +44 29 20874290

How to apply:

You can apply online - consideration is automatic on applying for a PhD in Biosciences

Please use our online application service at https://www.cardiff.ac.uk/study/postgraduate/research/programmes/programme/biosciences-phd-mphil-md

Please specify that you are applying for this particular project, the supervisor and source of funding.

Information on the application process can be found here

http://www.cardiff.ac.uk/study/postgraduate/applying

Entry requirements-

Applicants should have obtained, or be about to obtain, a first or upper second-class UK honours degree, or the equivalent qualification gained outside the UK, Applicants with a lower second class will only be considered if they also have a Master’s degree. Academic qualifications are considered alongside significant relevant non-academic experience.

English Language-

IELTS with an overall score of 6.5 with 5.5 in all subskills, or equivalent. Please see our English Language Requirements guidance for more details.