In‐vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE

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- teins (Cannon et al., 1989; Collier et al., 1988; Randall et al., 3990). Randall et al. (1986) suggested that there is a correlation between competence for export and lack of stable tertiary structure of precursors defined by protease resistance. It was shown by protease-resistance experiments (Collier et al., 1988), intrinsic-tryptophan-fluorescence spectroscopy (Liu et al., 1989) and immunoprecipitation studies (Weiss and Bassford, 1990) that SecB retards the folding of the precursor of the periplasmic maltose-binding protein (preMBP) and thereby maintains it in a translocation competent state. Additional studies on preMBP resulted in a model of kinetic partitioning between folding of the precursor a...

How proteins cross the bacterial cytoplasmic membrane

“…This view is supported by in vitro studies that demonstrate that precursor proteins can spontaneously form a translocation-competent state when diluted into solution from denaturant, while they lose their translocation competence during prolonged incubation in the absence of SecB [21,32,87,91]. SecB retards the folding of the precursor of maltose binding protein (pre-MBP) [126], and prevents the formation of insoluble aggregates of the rapidly folding precursors of the outer membrane proteins OmpA [86] and PhoE [21]. SecB associates with the mature domain of precursor proteins [ 126], but the specificity of this binding reaction has remained elusive.…”

“…SecB, however, stabilizes the unfolded protein in a soluble state, and hence may retard folding sufficiently to bring about a productive interaction of the precursor protein with other Sec proteins in order to become translocated. This view is supported by in vitro studies that demonstrate that precursor proteins can spontaneously form a translocation-competent state when diluted into solution from denaturant, while they lose their translocation competence during prolonged incubation in the absence of SecB [21,32,87,91]. SecB retards the folding of the precursor of maltose binding protein (pre-MBP) [126], and prevents the formation of insoluble aggregates of the rapidly folding precursors of the outer membrane proteins OmpA [86] and PhoE [21].…”

“…OmpA and PhoE bear a high amount of I]-structure both in their final conformations and in the SecB bound precursor forms [21,86]. SecB will also bind with low affinity to short peptides carrying a net positive charge (i.e., arginine and lysine residues) [124], including synthetic signal peptides (A.J.M.…”

A Thermodynamic Coupling Mechanism for the Disaggregation of a Model Peptide Substrate by Chaperone SecB

“…Since folded proteins are no longer competent for export, SecB binds to its substrates before complete folding has occurred and maintains them in an unfolded, translocation competent state (9,35). SecB has also been shown to prevent irreversible aggregation of two of its natural protein substrates OmpA and PhoE (6,36). SecB also prevents aggregation and promotes disaggregation of another natural substrate, maltose-binding protein.…”

Translocase-Bound SecA Is Largely Shielded from the Phospholipid Acyl Chains

“…in a translocation competent state by preventing aggregation and stable folding (19)(20)(21). Insertion of SecA is accompanied by the release of SecB into the cytosol (22) and a complete topological inversion of SecG (23).…”