title: Specific modulation of protein activity by using a bioorthogonal reaction.
abstract: Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use mutagenesis to insert an unnatural amino acid containing an azide group (Z) into the target protein at positions such that a "click" reaction with an alkyne modulator (X) will alter the function of the protein. This bioorthogonally reactive pair can engender specificity of X for the Z-containing protein, even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the Escherichia coli enzyme aminoacyl transferase by both active-site occlusion and allosteric mechanisms. We have termed this a "clickable magic bullet" strategy, and it should be generally applicable to studying the effects of protein inhibition, within the limits of unnatural amino acid mutagenesis.
authors.affiliation: Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323 (USA).
authors.forename: John B
authors.initials: JB
authors.lastname: Warner
authors.forename: Anand K
authors.initials: AK
authors.lastname: Muthusamy
authors.forename: E James
authors.initials: EJ
authors.lastname: Petersson
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D019881:Aminoacyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D057930:Click Chemistry; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D015202:Protein Engineering; D013329:Structure-Activity Relationship
chemical_list: D019881:Aminoacyltransferases
keywords: bioorthogonal chemistry; chemical genetics; click chemistry; covalent inhibitors; drug discovery; unnatural amino acids