title: Regulation of beta-lactamase activity by remote binding of heme: functional coupling of unrelated proteins through domain insertion.
abstract: Coupling the activities of normally disparate proteins into one functional unit has significant potential in terms of constructing novel switching components for synthetic biology or as biosensors. It also provides a means of investigating the basis behind transmission of conformation events between remote sites that is integral to many biological processes, including allostery. Here we describe how the structures and functions of two normally unlinked proteins, namely, the heme binding capability of cytochrome b(562) and the antibiotic degrading beta-lactamase activity of TEM, have been coupled using a directed evolution domain insertion approach. The important small biomolecule heme directly modulates in vivo and in vitro the beta-lactamase activity of selected integral fusion proteins. The presence of heme decreased the concentration of ampicillin tolerated by Escherichia coli and the level of in vitro hydrolysis of nitrocefin by up to 2 orders of magnitude. Variants with the largest switching magnitudes contained insertions at second-shell sites that abut key catalytic residues. Spectrophotometry confirmed that heme bound to the integral fusion proteins in a manner similar to that of cytochrome b(562). Circular dichroism suggested that only subtle structural changes rather than gross folding-unfolding events were responsible for modulating beta-lactamase activity, and size exclusion chromatography confirmed that the integral fusion proteins remained monomeric in both the apo and holo forms. Thus, by sampling a variety of insertion positions and linker sequences, we are able to couple the functions of two unrelated proteins by domain insertion.
authors.affiliation: School of Biosciences, Cardiff University, Cardiff, UK.
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Edwards
authors.forename: Abigail J
authors.initials: AJ
authors.lastname: Williams
authors.forename: Josephine L
authors.initials: JL
authors.lastname: Morris
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Baldwin
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
authors.forename: D Dafydd
authors.initials: DD
authors.lastname: Jones
mesh_terms: D000667:Ampicillin; D002511:Cephalosporins / Q000378:metabolism; D003573:Cytochrome b Group / Q000378:metabolism*; D019020:Directed Molecular Evolution; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006418:Heme / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013057:Spectrum Analysis; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D002511:Cephalosporins; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D006418:Heme; D000667:Ampicillin; C017850:cytochrome b562, E coli; D001618:beta-Lactamases; C021720:nitrocefin