Observing conformational and activity changes of tet repressor in vivo.
Effector triggered conformational changes of proteins such as regulators of transcription, receptors, or enzymes are the molecular basis for regulation in biology. Most proteins perform their biological functions intracellularly, in the presence of many potential interaction partners. Studies of conformational changes have mainly been performed in vitro using sophisticated physical and biochemical methods that usually require purified proteins. Here we describe the observation of conformational changes of Tet repressor in the cytoplasm of growing Escherichia coli cells, analyzed by ligand dependent disulfide crosslinking of cysteine residues substituted into mobile regions of the protein. The amount of protein undergoing the structural change is quantitatively linked to the concomitant induction of transcription of a reporter gene.
Tiebel
B
B
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany.
Garke
K
K
Hillen
W
W
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009875:Operator Regions, Genetic / Q000235:genetics; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013752:Tetracycline / Q000378:metabolism / Q000494:pharmacology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004220:Disulfides; D008024:Ligands; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline; D003545:Cysteine