Effect of Signal Peptide on Stability and Folding of Escherichia coli Thioredoxin

Pranveer Singh,S. Rajendra Kulothungan,Bharat V. Adkar,P. Shaik Syed Ali,Raghavan Varadarajan,Ravindra Singh Prajapati,Beena Krishnan,Likhesh Sharma,Rizwan H. Khan

doi:10.1371/journal.pone.0063442

Pranveer Singh, S. Rajendra Kulothungan + Show 7 more

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https://doi.org/10.1371/journal.pone.0063442

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Abstract

The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner.

Highlights

In E. coli two distinct pathways exist for the export of proteins across the cytoplasmic membrane
We explore the effects of two different signal peptides, pelB and malE on protein stability and aggregation in a smaller protein, E. coli thioredoxin. pelB refers to the 22 N-terminal leader sequence of pectatelyase B of Erwinia carotovora CE [26].The pelB leader sequence when attached to a protein, directs the protein to the bacterial periplasm, where the sequence is removed by a signal peptidase. pelB has been used to direct the coat protein-antigen fusions to the cell surface in engineered bacteriophages used for the purpose of phage display [27]
The data points are shown as closed circle ( ) for WT Trx, open circle (#) for pelB Trx and closed triangle (.) for malE Trx, all at 0.2 mg/ml concentration and open triangle (D) for malE Trx at a concentration of 2.5 mg/ ml (A) Representative differential scanning calorimetry (DSC) scans of WT Trx and pelB Trx, the line shows the fitting to a two state model with one peak. (B) Representative DSC scans of malE Trx at 0.2 mg/ml concentration, the line shows the fitting to non-two state model with a single peak.(C) Representative DSC scans of malE Trx at 2.5 mg/ml concentration; the line shows the fitting to a non-two-state with a single peak. doi:10.1371/journal.pone.0063442.g006

Summary

Introduction

In E. coli two distinct pathways exist for the export of proteins across the cytoplasmic membrane. Following interaction with the membrane receptor FtsY, the complex of nascent chain and ribosome is transferred to the SecYEG translocase In the latter post-translational pathway, upon emerging from the ribosome, proteins bind first to trigger factor, SecB and SecA. PelB has been used to direct the coat protein-antigen fusions to the cell surface in engineered bacteriophages used for the purpose of phage display [27] Both pelB and malE signal peptides utilize the post-translational translocation pathway. This has been experimentally shown for malE [28,29] and inferred for pelB based on the hydrophobicity of the signal sequence [27]. Measurements were done in CGH-10 buffer, pH 7.4 at 25uC with a 0.2 cm path-length cuvette. doi:10.1371/journal.pone.0063442.g002

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