Biochemistry in Bacterioferritin

Chemistry;Bacterioferritin;Encapsulation;Hexahistidine-affinity tag;Nickel-nitrilotriacetic acid;dynamic light scattering;gel filtration chromatography;fluorescence measurement

Suttisansanee, Uthaiwan

University of Waterloo

关键词: Chemistry;    Bacterioferritin;    Encapsulation;    Hexahistidine-affinity tag;    Nickel-nitrilotriacetic acid;    dynamic light scattering;    gel filtration chromatography;    fluorescence measurement;   

Others  :  https://uwspace.uwaterloo.ca/bitstream/10012/2983/1/usuttisa2006.pdf

瑞士|英语

来源: UWSPACE Waterloo Institutional Repository

【 摘 要 】

Bacterioferritin, an iron storage protein having a 24-subunit quaternary structure, was used as a model for the study of host-guest interactions and guest encapsulation, making use of its spherical cage-like structure.A hexahistidine-affinity tag fused to the C-terminus of each bacterioferritin subunit was constructed.The C-terminus of each subunit points toward the inside of the cavity, while the N-terminus is exposed on the surface of the protein.The hexaHistag was able to form strong interactions with a nickel-nitrilotriacetic acid linked dye molecule (guest) and this interaction was used in attempts to develop a principle to control guest molecule encapsulation within the spherical cavity of the 24-mer bacterioferritin protein molecule.The procedure involved (1) subunit dissociation under acidic pH, (2) affinity controlled dye-Histag binding with exposed C-terminal hexahistidine residues and (3) reassociation of the subunits at neutral pH.The encapsulation conditions involving step 1 and 3 were studied preliminarily using laser light scattering to measure size (hydrodynamic radius) of the protein particle with apoferritin as a model system as it resembles the size and structure of bacterioferritin.In order to encapsulate guest molecules, the emptied shell of bacterioferritin was generated by site-directed mutagenesis resulting in ferroxidase- as well as heme-free bacterioferritin mutants (E18A/M52L/E94A), and these mutants were used to examine protein stability before conducting encapsulation experiments.However, wild-type bacterioferritin possessed highest stability in maintaining its multisubunit structure; hence, it was used for the encapsulation studies.It was found that 100% bacterioferritin with hexahistidine tag at the C-terminus, and a combination of 60% bacterioferritin with hexahistidine tag at the C-terminus and 40% bacterioferritin without hexahistidine tag at the C-terminus yielded similar amounts of encapsulated guest molecules.This suggested that all hexahistidine at the C-terminus were not equally available for dye molecule binding.