title: Mutagenesis study to disrupt electrostatic interactions on the twofold symmetry interface of Escherichia coli bacterioferritin.
abstract: Ferritins and other cage proteins have been utilized as models to understand the fundamentals of protein folding and self-assembly. The bacterioferritin (BFR) from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for a mutagenesis study to investigate the role of electrostatic intermolecular interactions mediated through charged amino acids. Through structural and computational analyses, three charged amino acids R30, D56 and E60 which involved in an electrostatic interaction network were mutated to the opposite charge. Four mutants, R30D, D56R, E60H and D56R-E60H, were expressed, purified and characterized. All of the mutants fold into α-helical structures. Consistent with the computational prediction, they all show a lowered thermostability; double mutant D56R-E60H was found to be 16°C less stable than the wild type. Except for the mutant E60H, all the other mutations completely shut down the formation of protein cages to favour the dimer state in solution. The mutants, however, retain their ability to form cage-like nanostructures in the dried, surface immobilized conditions of transmission electron microscopy. Our findings confirm that even a single charge-inversion mutation at the 2-fold interface of BFR can affect the quaternary structure of its dimers and their ability to self-assemble into cage structures.
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China; Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, China;
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China;
authors.forename: Lijun
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Chemistry, King's College London, London, SE1 1DB, UK; and.
authors.forename: Maziar S
authors.initials: MS
authors.lastname: Ardejani
authors.affiliation: School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic 599489, Singapore.
authors.forename: Nur Fazlina
authors.initials: NF
authors.lastname: Aris
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China; Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, China;
authors.forename: Xun
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Chemistry, King's College London, London, SE1 1DB, UK; and.
authors.forename: Brendan P
authors.initials: BP
authors.lastname: Orner
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China; Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, China; hgwf@njfu.edu.cn.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D018698:Glutamic Acid / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D046912:Multiprotein Complexes / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D055503:Protein Multimerization / Q000235:genetics; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D055672:Static Electricity; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D008667:Metalloproteins; D046912:Multiprotein Complexes; C026121:bfr protein, E coli; D001224:Aspartic Acid; D018698:Glutamic Acid; D001120:Arginine
keywords: bacterioferritin; electrostatic interaction; nanocage; self-assembly; site-directed mutagenesis