期刊
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH
卷 1863, 期 12, 页码 3116-3124出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamcr.2016.09.006
关键词
Tat; Twin-Arginine Translocase; Signal peptide; Protein translocation
资金
- Biotechnology and Biological Sciences Research Council 'Bioprocessing Research Industry Club' grant [BB/K011219/1, BB/M006913/1]
- BBSRC [BB/L006391/1, BB/I017194/1, BB/M006913/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/M006913/1, BB/I017194/1, BB/L006391/1] Funding Source: researchfish
The Tat system transports folded proteins across the bacterial plasma membrane, and in Escherichia coli preferentially transports correctly-folded proteins. Little is known of the mechanism by which Tat proofreads a substrate's conformational state, and in this study we have addressed this question using a heterologous single-chain variable fragment (scFv) with a defined structure. We introduced mutations to surface residues while leaving the folded structure intact, and also tested the importance of conformational flexibility. We show that while the scFv is stably folded and active in the reduced form, formation of the 2 intra-domain disulphide bonds enhances Tat-dependent export 10-fold, indicating Tat senses the conformational flexibility and preferentially exports the more rigid structure. We further show that a 26-residue unstructured tail at the C-terminus blocks export, suggesting that even this short sequence can be sensed by the proofreading system. In contrast, the Tat system can tolerate significant changes in charge or hydrophobicity on the scFv surface; substitution of uncharged residues by up to 3 Lys-Glu pairs has little effect, as has the introduction of up to 5 Lys or Glu residues in a confined domain, or the introduction of a patch of 4 to 6 Leu residues in a hydrophilic region. We propose that the proofreading system has evolved to sense conformational flexibility and detect even very transiently-exposed internal regions, or the presence of unfolded peptide sections. In contrast, it tolerates major changes in surface charge or hydrophobicity. (C) 2016 Elsevier B.V. All rights reserved.
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