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LEAD SUPERVISOR: Professor Syma Khalid, Department of Biochemistry

Co-supervisor: Dr Philip Biggin, Department of Biochemistry

Commercial partner: Oxford Nanopore Technologies, Oxford

 

Introduction: Next generation DNA sequencing via nanopores pioneered by Oxford Nanopore Technologies uses an engineered version of the E. coli secretion lipoprotein CsgG. Electric-field driven movement of charged molecules through a nanopore embedded within an otherwise impermeable membrane lead to reductions in the signature current of that pore. The four bases of DNA each produce slightly different reductions, allowing them to be identified and thus the DNA to be sequenced.

There is great potential for this technology to be expanded to other biomedical applications such as in situ detection of toxins and viral particles and identifying post-translational modifications that lead to disease. This is directly within the MRC priority area of molecular and cellular medicine. Overall, given its inherent portability, with optimised speed and accuracy, nanopore based biosensing has the potential to transform detection and treatment of a range of diseases– the successes of DNA sequencing using this technology attest to its feasibility. To expand the scope of this technology to additional biosensing applications it is desirable to extend the repertoire of protein pores that may be used. The scaffold and mechanism of action of viral portal proteins have been identified as showing promise as potential pores. They are mechanically robust and are used by nature for molecular delivery. However, they can have some intrinsic gating of their own, which my interfere with the signal from the analyte and the biochemistry of the outer surface of these proteins prevents them being embedded into lipid bilayers, thereby reducing their usefulness.

Aims: We will use computational methods to predict routes to optimisation of several properties of viral portal proteins (initial focus on Phi29, others will be selected in consultation with ONT) for application as biosensors. This will build upon the expertise of the academic partner (Professor Syma Khalid, Biochemistry) and the industrial partner (Nanopore team at Oxford Nanopore Technologies Plc (ONT), led by Dr Jayne Wallace). Mutations and modifications to the proteins by the computational approach will be tested experimentally by ONT.

Methods:

Atomistic simulations to study the conformational dynamics of Phi29 portal complex (or similar system) to (a) identify any regions of mechanical weakness and intrinsic conformational lability and (b) to predict ways to optimise the complex based on the computational results combined with iterative refinement with feedback from experimental work by ONT.

Coarse-grained simulations to explore (a) how to modify the outer surface of the complex for embedding in various membranes and (b) membrane diffusion properties of the complex and how this may be manipulated.

Quantum mechanics to calculate the electrostatic profile of the designed proteins and determine how this would be impacted by analytes inside the pores. The most affected residues are likely to be the best candidates for modification/mutation.

Recent advancements in protein structure prediction such as AlphaFold2 provide additional tools to facilitate the optimisation of protein-nanopores and make this project extremely timely.

Outputs: By the end of the PhD, we will have:

(a)   Developed a computational pipeline for optimisation of a given protein pore for application as a biosensor.

(b)   Published at least 3 papers.

 

Apply using course: DPhil in Biochemistry

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