Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol.

Details

Serval ID
serval:BIB_5A2ABAB8B9CC
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol.
Journal
Scientific reports
Author(s)
Tavares D., Reimer A., Roy S., Joublin A., Sentchilo V., van der Meer J.R.
ISSN
2045-2322 (Electronic)
ISSN-L
2045-2322
Publication state
Published
Issued date
15/11/2019
Peer-reviewed
Oui
Volume
9
Number
1
Pages
16940
Language
english
Notes
Publication types: Journal Article
Publication Status: epublish
Abstract
Bacterial periplasmic-binding proteins have been acclaimed as general biosensing platform, but their range of natural ligands is too limited for optimal development of chemical compound detection. Computational redesign of the ligand-binding pocket of periplasmic-binding proteins may yield variants with new properties, but, despite earlier claims, genuine changes of specificity to non-natural ligands have so far not been achieved. In order to better understand the reasons of such limited success, we revisited here the Escherichia coli RbsB ribose-binding protein, aiming to achieve perceptible transition from ribose to structurally related chemical ligands 1,3-cyclohexanediol and cyclohexanol. Combinations of mutations were computationally predicted for nine residues in the RbsB binding pocket, then synthesized and tested in an E. coli reporter chassis. Two million variants were screened in a microcolony-in-bead fluorescence-assisted sorting procedure, which yielded six mutants no longer responsive to ribose but with 1.2-1.5 times induction in presence of 1 mM 1,3-cyclohexanediol, one of which responded to cyclohexanol as well. Isothermal microcalorimetry confirmed 1,3-cyclohexanediol binding, although only two mutant proteins were sufficiently stable upon purification. Circular dichroism spectroscopy indicated discernable structural differences between these two mutant proteins and wild-type RbsB. This and further quantification of periplasmic-space abundance suggested most mutants to be prone to misfolding and/or with defects in translocation compared to wild-type. Our results thus affirm that computational design and library screening can yield RbsB mutants with recognition of non-natural but structurally similar ligands. The inherent arisal of protein instability or misfolding concomitant with designed altered ligand-binding pockets should be overcome by new experimental strategies or by improved future protein design algorithms.
Pubmed
Web of science
Open Access
Yes
Create date
29/11/2019 21:40
Last modification date
16/07/2020 9:43
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