title: Exploring intermolecular interactions of a substrate binding protein using a riboswitch-based sensor.
abstract: The study of biological transporters can be hampered by a dearth of methodology for tracking their activity within cells. Here, we present a means of monitoring the function of transport machinery within bacteria, exploiting a genetically encoded riboswitch-based sensor to detect the accumulation of the substrate in the cytoplasm. This method was used to investigate the model ABC transporter BtuC2D2F, which permits vitamin B12 uptake in Escherichia coli. We exploited the wealth of structural data available for this transporter to probe the functional and mechanistic importance of key residues of the substrate binding protein BtuF that are predicted to support its interaction with its substrate or with the BtuC channel-forming subunits. Our results reveal molecular interaction requirements for substrate binding proteins and demonstrate the utility of riboswitch-based sensors in the study of biological transport.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada.
authors.forename: Casey C
authors.initials: CC
authors.lastname: Fowler
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada.
authors.forename: Seiji
authors.initials: S
authors.lastname: Sugiman-Marangos
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada.
authors.forename: Murray S
authors.initials: MS
authors.lastname: Junop
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada; Michael G. DeGroote Institute of Infectious Disease Research, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Brown
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada; Michael G. DeGroote Institute of Infectious Disease Research, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada; Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada. Electronic address: liying@mcmaster.ca.
authors.forename: Yingfu
authors.initials: Y
authors.lastname: Li
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D058928:Riboswitch; D013379:Substrate Specificity; D014805:Vitamin B 12 / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; D058928:Riboswitch; C446048:btuF protein, E coli; D014805:Vitamin B 12