Three-State Equilibrium of Escherichia coli Trigger Factor
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H. Patzelt
Abstract
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 ribosomeassociated after cell lysis. Here we show that TF follows a threestate equilibrium. Size exclusion chromatography, crosslinking and analytical ultracentrifugation revealed that uncomplexed TF dimerizes with an apparent Kd of 18 M. Dimerization is mediated by the Nterminal ribosome binding domain and the Cterminal domain of TF, whereas the central peptidyl prolyl isomerase (PPIase) and substrate binding domain does not contribute to dimerization. Crosslinking experiments showed that TF is monomeric in its ribosomeassociated state. Quantitative analysis of TF binding to ribosomes revealed a dissociation constant for the TFribosome complex of approximately 1.2 M. From these data we estimate that in vivo most of the ribosomes are in complex with monomeric TF. Uncomplexed TF, however, is in a monomerdimer equilibrium with approximately two thirds of TF existing in a dimeric state.
Copyright © 2002 by Walter de Gruyter GmbH & Co. KG
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