title: Sequence context and crosslinking mechanism affect the efficiency of in vivo capture of a protein-protein interaction.
abstract: Protein-protein interactions (PPIs) are essential for implementing cellular processes and thus methods for the discovery and study of PPIs are highly desirable. An emerging method for capturing PPIs in their native cellular environment is in vivo covalent chemical capture, a method that uses nonsense suppression to site specifically incorporate photoactivable unnatural amino acids (UAAs) in living cells. However, in one study we found that this method did not capture a PPI for which there was abundant functional evidence, a complex formed between the transcriptional activator Gal4 and its repressor protein Gal80. Here we describe the factors that influence the success of covalent chemical capture and show that the innate reactivity of the two UAAs utilized, (p-benzoylphenylalanine (pBpa) and p-azidophenylalanine (pAzpa)), plays a profound role in the capture of Gal80 by Gal4. Based upon these data, guidelines are outlined for the successful use of in vivo photo-crosslinking to capture novel PPIs and to characterize the interfaces.
authors.affiliation: Program in Chemical Biology, University of Michigan, Ann Arbor, MI, 48109.
authors.forename: Jody K
authors.initials: JK
authors.lastname: Lancia
authors.forename: Adaora
authors.initials: A
authors.lastname: Nwokoye
authors.forename: Amanda
authors.initials: A
authors.lastname: Dugan
authors.forename: Cassandra
authors.initials: C
authors.lastname: Joiner
authors.forename: Rachel
authors.initials: R
authors.lastname: Pricer
authors.forename: Anna K
authors.initials: AK
authors.lastname: Mapp
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism; D001577:Benzophenones / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D000099160:LexA Repressor Protein
chemical_list: D000596:Amino Acids; D001386:Azides; D001426:Bacterial Proteins; D001577:Benzophenones; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D008715:Methionine; D050505:Mutant Proteins; D010649:Phenylalanine; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein; C488060:4-benzoylphenylalanine; C052130:GAL4 protein, S cerevisiae; C027162:4-azidophenylalanine
keywords: Gal4; Gal80; crosslinking; protein-protein interactions; unnatural amino acid
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