title: An expanded genetic code in Candida albicans to study protein-protein interactions in vivo.
abstract: For novel insights into the pathogenicity of Candida albicans, studies on molecular interactions of central virulence factors are crucial. Since methods for the analysis of direct molecular interactions of proteins in vivo are scarce, we expanded the genetic code of C. albicans with the synthetic photo-cross-linking amino acid p-azido-L-phenylalanine (AzF). Interacting molecules in close proximity of this unnatural amino acid can be covalently linked by UV-induced photo-cross-link, which makes unknown interacting molecules available for downstream identification. Therefore, we applied an aminoacyl-tRNA synthetase and a suppressor tRNA pair (EcTyrtRNA(CUA)) derived from Escherichia coli, which was previously reported to be orthogonal in Saccharomyces cerevisiae. We further optimized the aminoacyl-tRNA synthetase for AzF (AzF-RS) and EcTyrtRNA(CUA) for C. albicans and identified one AzF-RS with highest charging efficiency. Accordingly, incorporation of AzF into selected model proteins such as Tsa1p or Tup1p could be considerably enhanced. Immunologic detection of C-terminally tagged Tsa1p and Tup1p upon UV irradiation in a strain background containing suppressor tRNA and optimized AzF-RS revealed not only the mutant monomeric forms of these proteins but also higher-molecular-weight complexes, strictly depending on the specific position of incorporated AzF and UV excitation. By Western blotting and tandem mass spectrometry, we could identify these higher-molecular-weight complexes as homodimers consisting of one mutant monomer and a differently tagged, wild-type version of Tsa1p or Tup1p, respectively, demonstrating that expanding the genetic code of C. albicans with the unnatural photo-cross-linker amino acid AzF and applying it for in vivo binary protein interaction analyses is feasible.
authors.affiliation: IGVT, University of Stuttgart, Stuttgart, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Palzer
authors.forename: Yannick
authors.initials: Y
authors.lastname: Bantel
authors.forename: Franziska
authors.initials: F
authors.lastname: Kazenwadel
authors.forename: Michael
authors.initials: M
authors.lastname: Berg
authors.forename: Steffen
authors.initials: S
authors.lastname: Rupp
authors.forename: Kai
authors.initials: K
authors.lastname: Sohn
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002176:Candida albicans / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005815:Genetic Code; D008958:Models, Molecular; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000235:genetics* / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D001386:Azides; D001426:Bacterial Proteins; D005656:Fungal Proteins; D012346:RNA, Transfer, Amino Acyl; D012097:Repressor Proteins; C038012:tRNA, azidophenylalanine-; D010649:Phenylalanine; D010544:Peroxidases; D000604:Amino Acyl-tRNA Synthetases
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