title: DNA sequencing with MspA: Molecular Dynamics simulations reveal free-energy differences between sequencing and non-sequencing mutants.
abstract: MspA has been identified as a promising candidate protein as a component of a nanopore-based DNA-sequencing device. However the wildtype protein must be engineered to incorporate all of the features desirable for an accurate and efficient device. In the present study we have utilized atomistic molecular dynamics to perform umbrella-sampling calculations to calculate the potential of mean force (PMF) profiles for translocation of the four DNA nucleotides through MspA. We show there is an energetic barrier to translocation of individual nucleotides through a mutant that closely resembles the wildtype protein, but not through a mutant engineered for the purpose of sequencing. Crucially we are able to quantify the change in free energy for mutating key residues. Thus providing a quantitative characterisation of the energetic impact of individual amino acid sidechains on nucleotide translocation through the pore of MspA.
authors.affiliation: Chemistry, Faculty of Natural and Environmental Sciences, University of Southampton, Southampton, SO17 1BJ, United Kingdom.
authors.forename: Richard M A
authors.initials: RM
authors.lastname: Manara
authors.affiliation: Oxford Nanopore Technologies Ltd, Oxford United Kingdom.
authors.forename: E Jayne
authors.initials: EJ
authors.lastname: Wallace
authors.affiliation: Chemistry, Faculty of Natural and Environmental Sciences, University of Southampton, Southampton, SO17 1BJ, United Kingdom.
authors.forename: Syma
authors.initials: S
authors.lastname: Khalid
mesh_terms: D004247:DNA / Q000737:chemistry*; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D009154:Mutation; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D013816:Thermodynamics
chemical_list: D018272:Porins; C121838:mspA protein, Mycobacterium smegmatis; D004247:DNA