Three-state equilibrium of Escherichia coli trigger factor.
Trigger Factor (TF) is the first chaperone that interacts with nascent chains of cytosolic proteins in Escherichia coli. Although its chaperone activity requires association with ribosomes, TF is present in vivo in a 2-3 fold molar excess over ribosomes and a fraction of it is not ribosome-associated after cell lysis. Here we show that TF follows a three-state equilibrium. Size exclusion chromatography, crosslinking and analytical ultracentrifugation revealed that uncomplexed TF dimerizes with an apparent Kd of 18 microM. Dimerization is mediated by the N-terminal ribosome binding domain and the C-terminal domain of TF, whereas the central peptidyl prolyl isomerase (PPlase) and substrate binding domain does not contribute to dimerization. Crosslinking experiments showed that TF is monomeric in its ribosome-associated state. Quantitative analysis of TF binding to ribosomes revealed a dissociation constant for the TF-ribosome complex of approximately 1.2 microM. From these data we estimate that in vivo most of the ribosomes are in complex with monomeric TF. Uncomplexed TF, however, is in a monomer-dimer equilibrium with approximately two thirds of TF existing in a dimeric state.
Patzelt
Holger
H
Zentrum für Molekulare Biologie (ZMBH), Universität Heidelberg, INF282, D-69120 Heidelberg, Germany.
Kramer
Günter
G
Rauch
Thomas
T
Schönfeld
Hans-Joachim
HJ
Bukau
Bernd
B
Deuerling
Elke
E
D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005976:Glutaral / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000378:metabolism; D014461:Ultracentrifugation / Q000379:methods
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase; D005976:Glutaral