Biophysical studies of biological nanopores used for biopolymer sequencing
File(s)
Author(s)
Glencross, Flo
Type
Thesis
Abstract
Nanopore sequencing (NS) has emerged in the past decade, as a forerunner for accurate, inexpensive, long-read biopolymer sequencing. Biological pores, inserted into synthetic membranes, are placed under applied voltages allowing them to translocate DNA/RNA. Distinct alterations in a measured ionic current can be assigned to specific nucleotides, allowing the biopolymer to be sequenced. The accuracy of this method is founded in the dimensions and hydropathy of the smallest aperture of the pore, termed the ‘constriction’ and these properties can be manipulated through point mutations in the protein sequence. Current commercial protein engineering of NS pores involves random mutagenesis, coupled with electrophysiology experiments, to determine sequencing ability, which can be expensive and time consuming. This approach can be used to infer and build on favourable geometry and chemical properties of the pore constriction, however the interactions between the protein and DNA are still poorly understood and likely influenced by protein dynamics.
This research attempts to characterise the dynamic nature of the derivative mutants of the nanopores CsgG and lysenin, through experimental and computational studies. Initial work focuses on optimisation of the expression and purification of these proteins for solution NMR experiments, however insufficient yield of both pores made this unfeasible. Molecular dynamics (MD) was then utilised to investigate the behaviour of wild-type (WT) CsgG under the NS conditions, involving the simulation of an electric field and the presence of ssDNA. From this, the constriction was identified to have increased flexibility and form the most interactions with DNA through specific residues, identifying these as potential points for mutagenesis. Using an in-silico rational design software called ‘PoreDesigner’, WT CsgG was mutated in the constriction to varying the dimensions and hydropathy, altering predicted DNA interactions. Electrophysiology measurements and MD studies on CsgG determined increased hydrophobicity to be a favourable property for biological NS pores and developed a potential high-throughput method for rational design.
This research attempts to characterise the dynamic nature of the derivative mutants of the nanopores CsgG and lysenin, through experimental and computational studies. Initial work focuses on optimisation of the expression and purification of these proteins for solution NMR experiments, however insufficient yield of both pores made this unfeasible. Molecular dynamics (MD) was then utilised to investigate the behaviour of wild-type (WT) CsgG under the NS conditions, involving the simulation of an electric field and the presence of ssDNA. From this, the constriction was identified to have increased flexibility and form the most interactions with DNA through specific residues, identifying these as potential points for mutagenesis. Using an in-silico rational design software called ‘PoreDesigner’, WT CsgG was mutated in the constriction to varying the dimensions and hydropathy, altering predicted DNA interactions. Electrophysiology measurements and MD studies on CsgG determined increased hydrophobicity to be a favourable property for biological NS pores and developed a potential high-throughput method for rational design.
Version
Open Access
Date Issued
2021-06
Date Awarded
2021-11
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Matthews, Stephen
Rouse, Sarah
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)