In-vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE. Evidence that SecB prevents the precursor from aggregating by forming A functional complex (original) (raw)

1992, European Journal of Biochemistry

Abstract

We characterised the behaviour of the purified precursor protein prePhoE upon dilution from 8 M urea by CD, fluorescence spectroscopy and gel-filtration techniques. It is demonstrated that prePhoE rapidly adopts j3 structure, folds and aggregates upon dilution to urea concentrations below 3 M. These processes are paralleled by a loss of translocation competence. Furthermore the interaction of prePhoE with SecB was investigated. SecB is shown to have a very high content of fi structure, therefore we propose that precursor recognition by SecB is mediated through p-fl interaction. It is shown that SecB has little effect on the adoption of secondary structure and tertiary folding upon dilution of the precursor from urea. However, SecB prevents the precursor from aggregating by forming a functional and stable complex Escherichia coli periplasmic and outer-membrane proteins are synthesized in the cytosol as precursors carrying an Nterminal signal sequence and are subsequently translocated across the inner membrane, requiring both cytosolic and membrane factors. SecB is one of the cytosolic factors which is required for the translocation of a subset of precursor proteins (Kumamoto and Beckwith, 1985). In-vitro studies with SecB purified from an over-producing (Weiss et al., 1988; Kumamoto et al., 1989) or non-over-producing (Watanabe and Blobel, 1989a) strain, showed that it promotes in a tetrameric (Watanabe and Blobel, 1989a) or an oligomeric (Weiss et al., 1988) form the translocation of in-vitrosynthesized precursor proteins. It has also been shown that SecB stabilizes the translocation-competent state of the purified precursors of the E. coli outer membrane proteins PhoE (prePhoE) and OmpA (preOmpA) diluted from concentrated urea solutions in vitro (Kusters et al., 1989; Lecker et al., 1989). Two major hypotheses are popular on the nature of the interaction between SecB and precursor proteins. In one it is believed that SecB acts in targeting, as a prokaryotic equivalent of the signal-recognition particle by binding to the signal sequence of precursor proteins prior to their translocation (Watanabe and Blobel, 1989b). The other hypothesis is that SecB acts as a chaperone keeping precursor proteins in a translocation-competent state by retarding their folding, through binding to the mature part of the precursor pro-Correspondence to E. Breukink, Centre for Biomembranes and

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