scholarly journals Computational design of ligand-binding proteins with high affinity and selectivity

Nature ◽  
2013 ◽  
Vol 501 (7466) ◽  
pp. 212-216 ◽  
Author(s):  
Christine E. Tinberg ◽  
Sagar D. Khare ◽  
Jiayi Dou ◽  
Lindsey Doyle ◽  
Jorgen W. Nelson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Proteins ◽  
Computational Design ◽  
High Affinity

scholarly journals Computational redesign of theEscherichia coliribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol

2019 ◽  
Author(s):  
Diogo Tavares ◽  
Artur Reimer ◽  
Shantanu Roy ◽  
Aurélie Joublin ◽  
Vladimir Sentchilo ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Proteins ◽  
Binding Protein ◽  
Computational Design ◽  
Binding Pocket ◽  
Wild Type ◽  
Ligand Binding Pocket ◽  
Mutant Proteins ◽  
Periplasmic Binding Proteins ◽  
Natural Ligands

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 theEscherichia coliRbsB 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 anE. colireporter 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.

Atomic structures of periplasmic binding proteins and the high-affinity active transport systems in bacteria

1990 ◽  
Vol 326 (1236) ◽  
pp. 341-352 ◽  
Keyword(s):  
Ligand Binding ◽  
Active Transport ◽  
Binding Proteins ◽  
Osmotic Shock ◽  
Transport Systems ◽  
Binding Studies ◽  
Tertiary Structures ◽  
Atomic Structures ◽  
High Affinity ◽  
Periplasmic Binding Proteins

We have determined and refined the X-ray crystal structures of six periplasmic binding proteins that serve as initial receptors for the osmotic-shock sensitive, active transport of L-arabinose, D-galactose/D-glucose, maltose, sulphate, leucine/isoleucine/ valine and leucine. The tertiary structures and atomic interactions between proteins and ligands show common features that are important for understanding the function of the binding proteins. All six structures are ellipsoidal, consisting of two similar, globular domains. The ligand-binding site is located deep in the cleft between the two domains. Irrespective of the nature of the ligand (e.g. saccharide, sulphate dianion or leucine zwitterion), the specificities and affinities of the binding sites are achieved mainly through hydrogen-bonding interactions. Binding of ligands induces a large protein conformational change. Three different structures have been observed among the binding proteins: unliganded ‘open cleft´, liganded ‘open cleft’, and liganded ‘closed cleft5. Here we discuss the functions of binding proteins in the light of numerous crystallographic and ligand-binding studies and propose a mechanism for the binding protein-dependent, high-affinity active transport.

Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS)

10.1038/89281 ◽  
2001 ◽  
Vol 19 (6) ◽  
pp. 537-542 ◽  
Author(s):  
Gang Chen ◽  
Andrew Hayhurst ◽  
Jeffery G. Thomas ◽  
Barrett R. Harvey ◽  
Brent L. Iverson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Proteins ◽  
Periplasmic Expression ◽  
High Affinity ◽  
Affinity Ligand ◽  
High Affinity Ligand
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