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Abstract
Proteins must be unfolded in order to be translocated across the cytoplasmic membrane by the Sec translocation pathway in Escherichia coli. Most cleavable, N-terminal signal sequences in E. coli (such as the PhoA signal sequence) promote posttranslational translocation. Signal sequences that promote posttranslational translocation cannot promote efficient translocation of the normally cytoplasmic protein thioredoxin-1 to the periplasm because thioredoxin rapidly folds before it can be translocated and becomes trapped in the cytoplasm. I isolated a collection of thioredoxin folding mutants that can be more efficiently translocated to the periplasm by the posttranslational pathway by selecting for increased motility in a dsbA mutant. I purified a representative set of these thioredoxin mutants and assayed their folding in vitro. All but one of the mutant proteins assayed have kinetic and thermodynamic folding defects in vitro. The amino acid substitution that causes the largest folding defect, a leucine to a proline change in the penultimate amino acid, would not have been predicted a priori to have such a significant effect on protein folding.
Recent work demonstrated that one native E. coli signal sequence, the DsbA signal sequence, can promote efficient translocation of thioredoxin to the periplasm. This efficient translocation is dependent on the signal recognition particle (SRP) and appears to be cotranslational. I used an iterative approach to screen for signal sequences that promote efficient SRP-dependent translocation of thioredoxin. I identified a subset of E. coli signal sequence that promote cotranslational, SRP-dependent translocation and developed a computer program that can predict whether a signal sequence is likely to promote SRP-dependent cotranslational translocation. An analysis of the signal sequences suggests that the SRP recognizes ∼10% of all E. coli signal sequences based on hydrophobicity in addition to some as yet undiscovered property. In addition, I showed that the DsbA protein requires an SRP-dependent signal sequence to be efficiently translocated to the periplasm. This is the first example in which the signal sequences of normally exported proteins cannot be freely exchanged without a translocation defect. The work presented in this dissertation demonstrates the intimate connection between protein folding and protein translocation in E. coli.
