title: Intein-mediated cyclization of bacterial acyl carrier protein stabilizes its folded conformation but does not abolish function.
abstract: Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Volkmann
authors.forename: Peter W
authors.initials: PW
authors.lastname: Murphy
authors.forename: Elden E
authors.initials: EE
authors.lastname: Rowland
authors.forename: John E
authors.initials: JE
authors.lastname: Cronan
authors.forename: Xiang-Qin
authors.initials: XQ
authors.lastname: Liu
authors.forename: Christian
authors.initials: C
authors.lastname: Blouin
authors.forename: David M
authors.initials: DM
authors.lastname: Byers
mesh_terms: D000213:Acyl Carrier Protein / Q000737:chemistry*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D047668:Inteins; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D053719:Tandem Mass Spectrometry / Q000379:methods; D014733:Vibrio / Q000378:metabolism
chemical_list: D000213:Acyl Carrier Protein; D010743:Phospholipids