title: Site-specific modification of Candida antarctica lipase B via residue-specific incorporation of a non-canonical amino acid.
abstract: In order to modify proteins in a controlled way, new functionalities need to be introduced in a defined manner. One way to accomplish this is by the incorporation of a non-natural amino acid of which the side chain can selectively be reacted to other molecules. We have investigated whether the relatively simple method of residue-specific replacement of methionine by azidohomoalanine can be used to achieve monofunctionalization of the model enzyme Candida antarctica lipase B. A protein variant was engineered with one additional methionine residue. Due to the high hydrophobicity and low abundance of methionine, this was the only residue out of five that was exposed to the solvent. The use of the Cu (I)-catalyzed [3 + 2] cycloaddition under native conditions resulted in a monofunctionalized enzyme which retained hydrolytic activity. The strategy can be considered a convenient tool to modify proteins at a single position as long as one solvent-exposed methionine is available.
authors.affiliation: Department of Bioorganic Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, The Netherlands.
authors.forename: Sanne
authors.initials: S
authors.lastname: Schoffelen
authors.forename: Mark H L
authors.initials: MH
authors.lastname: Lambermon
authors.forename: Mark B
authors.initials: MB
authors.lastname: van Eldijk
authors.forename: Jan C M
authors.initials: JC
authors.lastname: van Hest
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites; D002175:Candida / Q000201:enzymology*; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D008049:Lipase