title: In Vivo Photo-Cross-Linking to Study T3S Interactions Demonstrated Using the Yersinia pestis T3S System.
abstract: Cross-linking of proteins is effective in determining protein-protein interactions. The use of photo-cross-linkers was developed to study protein interactions in several manners. One method involved the incorporation of photo-activatable cross-linking groups into chemically synthesized peptides. A second approach relies on incorporation of photo-activatable cross-linking groups into proteins using tRNAs with chemically bound photo-activatable amino acids with suppressor tRNAs translational systems to incorporate the tags into specific sites. A third system was made possible by the development of photoreactive amino acids that use the normal cellular tRNAs and aminoacyl tRNA synthetases. In this method, the third system is used to demonstrate its utility for the study of T3S system interactions. This method describes how two photo-activatable amino acids, photo-methionine and photo-leucine, that use the normal cellular machinery are incorporated into Yersinia pestis and used to study interactions in the T3S system. To demonstrate the system, the method was used to cross-link the T3S regulatory proteins LcrG and LcrV.
authors.affiliation: Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, 1301 North Columbia Road, Stop 9037, Grand Forks, ND, 58203, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Henderson
authors.affiliation: Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, USA. matthew.nilles@med.und.edu.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Nilles
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002846:Chromatography, Affinity; D007930:Leucine / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D000069296:Type III Secretion Systems / Q000235:genetics / Q000378:metabolism*; D015010:Yersinia pestis / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D052899:Pore Forming Cytotoxic Proteins; D000069296:Type III Secretion Systems; D008715:Methionine; D007930:Leucine
keywords: LcrG; LcrV; Photo-cross-linking; Photo-leucine; Photo-methionine; Type III secretion system (T3SS); Yersinia pestis