Mechanical Unfolding of Ribose Binding Protein and Its Comparison with Other Periplasmic Binding Proteins

2014 ◽  
Vol 118 (39) ◽  
pp. 11449-11454 ◽  
Author(s):  
Hema Chandra Kotamarthi ◽  
Satya Narayan ◽  
Sri Rama Koti Ainavarapu
Keyword(s):  
Binding Proteins ◽  
Binding Protein ◽  
Periplasmic Binding Proteins ◽  
Ribose Binding Protein

Subcellular localization defects characterize ribose-binding mutant proteins with new ligand properties in Escherichia coli

2021 ◽  
Author(s):  
Diogo Tavares ◽  
Jan R. van der Meer
Keyword(s):  
Escherichia Coli ◽  
Subcellular Localization ◽  
Ligand Binding ◽  
Binding Proteins ◽  
Binding Protein ◽  
Binding Pocket ◽  
Binding Properties ◽  
General Properties ◽  
Periplasmic Binding Proteins ◽  
Ribose Binding Protein

Periplasmic-binding proteins have been previously proclaimed as a general scaffold to design sensor proteins with new recognition specificities for non-natural compounds. Such proteins can be integrated in bacterial bioreporter chassis with hybrid chemoreceptors to produce a concentration-dependent signal after ligand binding to the sensor cell. However, computationally designed new ligand-binding properties ignore the more general properties of periplasmic binding proteins, such as their periplasmic translocation, dynamic transition of open and closed forms, and interactions with membrane receptors. In order to better understand the roles of such general properties in periplasmic signaling behaviour, we study here the subcellular localization of ribose-binding protein (RbsB) in Escherichia coli in comparison to a recently evolved set of mutants designed to bind 1,3-cyclohexanediol. As proxies for localization we calibrate and deploy C-terminal end mCherry fluorescent protein fusions. Whereas RbsB-mCherry coherently localized to the periplasmic space and accumulated in (periplasmic) polar regions depending on chemoreceptor availability, mutant RbsB-mCherry expression resulted in high fluorescence cell-to-cell variability. This resulted in higher proportions of cells devoid of clear polar foci and of cells with multiple fluorescent foci elsewhere, suggesting poorer translocation, periplasmic autoaggregation and mislocalization. Analysis of RbsB mutants and mutant libraries at different stages of directed evolution suggested overall improvement to more RbsB-wild-type-like characteristics, which was corroborated by structure predictions. Our results show that defects in periplasmic localization of mutant RbsB proteins partly explains their poor sensing performance. Future efforts should be directed to predicting or selecting secondary mutations outside computationally designed binding pockets that take folding, translocation and receptor-interactions into account. Importance Biosensor engineering relies on transcription factors or signaling proteins to provide the actual sensory functions for the target chemicals. Since for many compounds there are no natural sensory proteins, there is a general interest in methods that could unlock routes to obtaining new ligand-binding properties. Bacterial periplasmic-binding proteins (PBPs) form an interesting family of proteins to explore to this purpose, because there is a large natural variety suggesting evolutionary trajectories to bind new ligands. PBPs are conserved and amenable to accurate computational binding pocket predictions. However, studying ribose-binding protein in Escherichia coli we discovered that designed variants have defects in their proper localization in the cell, which can impair appropriate sensor signaling. This indicates that functional sensing capacity of PBPs cannot be obtained solely through computational design of the ligand-binding pocket, but must take other properties of the protein into account, which are currently very difficult to predict.

scholarly journals Ribose-Binding Protein Mutants With Improved Interaction Towards the Non-natural Ligand 1,3-Cyclohexanediol

2021 ◽  
Vol 9 ◽  
Author(s):  
Diogo Tavares ◽  
Jan Roelof van der Meer
Keyword(s):  
Ligand Binding ◽  
Binding Proteins ◽  
Binding Protein ◽  
Binding Pocket ◽  
Ligand Binding Pocket ◽  
Natural Ligand ◽  
Protein Mutants ◽  
Periplasmic Binding Proteins ◽  
Natural Ligands ◽  
Ribose Binding Protein

Bioreporters consist of genetically modified living organisms that respond to the presence of target chemical compounds by production of an easily measurable signal. The central element in a bioreporter is a sensory protein or aptamer, which, upon ligand binding, modifies expression of the reporter signal protein. A variety of naturally occurring or modified versions of sensory elements has been exploited, but it has proven to be challenging to generate elements that recognize non-natural ligands. Bacterial periplasmic binding proteins have been proposed as a general scaffold to design receptor proteins for non-natural ligands, but despite various efforts, with only limited success. Here, we show how combinations of randomized mutagenesis and reporter screening improved the performance of a set of mutants in the ribose binding protein (RbsB) of Escherichia coli, which had been designed based on computational simulations to bind the non-natural ligand 1,3-cyclohexanediol (13CHD). Randomized mutant libraries were constructed that used the initially designed mutants as scaffolds, which were cloned in an appropriate E. coli bioreporter system and screened for improved induction of the GFPmut2 reporter fluorescence in presence of 1,3-cyclohexanediol. Multiple rounds of library screening, sorting, renewed mutagenesis and screening resulted in 4.5-fold improvement of the response to 1,3-cyclohexanediol and a lower detection limit of 0.25 mM. All observed mutations except one were located outside the direct ligand-binding pocket, suggesting they were compensatory and helping protein folding or functional behavior other than interaction with the ligand. Our results thus demonstrate that combinations of ligand-binding-pocket redesign and randomized mutagenesis can indeed lead to the selection and recovery of periplasmic-binding protein mutants with non-natural compound recognition. However, current lack of understanding of the intermolecular movement and ligand-binding in periplasmic binding proteins such as RbsB are limiting the rational production of further and better sensory mutants.

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

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Diogo Tavares ◽  
Artur Reimer ◽  
Shantanu Roy ◽  
Aurélie Joublin ◽  
Vladimir Sentchilo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Binding Proteins ◽  
Binding Pocket ◽  
Wild Type ◽  
Ligand Binding Pocket ◽  
Mutant Proteins ◽  
Periplasmic Binding Proteins ◽  
Natural Ligands ◽  
Ribose Binding Protein

AbstractBacterial 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.

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.

scholarly journals Ligand-Free Open−Closed Transitions of Periplasmic Binding Proteins: The Case of Glutamine-Binding Protein

Biochemistry ◽  
2010 ◽  
Vol 49 (9) ◽  
pp. 1893-1902 ◽  
Author(s):  
Guillermo A. Bermejo ◽  
Marie-Paule Strub ◽  
Chien Ho ◽  
Nico Tjandra
Keyword(s):  
Binding Proteins ◽  
Binding Protein ◽  
Periplasmic Binding Proteins ◽  
Ligand Free

scholarly journals Holo- and Apo-bound Structures of Bacterial Periplasmic Heme-binding Proteins

2007 ◽  
Vol 282 (49) ◽  
pp. 35796-35802 ◽  
Author(s):  
Winny W. Ho ◽  
Huiying Li ◽  
Suntara Eakanunkul ◽  
Yong Tong ◽  
Angela Wilks ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Binding Protein ◽  
Heme Iron ◽  
Shigella Dysenteriae ◽  
Class Iii ◽  
Heme Pocket ◽  
Heme Transport ◽  
Binding Domains ◽  
Periplasmic Binding Proteins ◽  
Common Architecture

An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an “in” position where it can coordinate the heme iron to an “out” orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg228 in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg228, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B12-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B12, compared with ligands for FhuD, a peptide siderophore.

scholarly journals Periplasmic Binding Proteins in Thermophiles: Characterization and Potential Application of an Arginine-Binding Protein from Thermotoga maritima: A Brief Thermo-Story

Life ◽  
2013 ◽  
Vol 3 (1) ◽  
pp. 149-160 ◽  
Author(s):  
Alessio Ausili ◽  
Maria Staiano ◽  
Jonathan Dattelbaum ◽  
Antonio Varriale ◽  
Alessandro Capo ◽  
...  
Keyword(s):  
Potential Application ◽  
Binding Proteins ◽  
Thermotoga Maritima ◽  
Binding Protein ◽  
Periplasmic Binding Proteins

scholarly journals Phosphorylation of the Periplasmic Binding Protein in Two Transport Systems for Arginine Incorporation in Escherichia coli K-12 Is Unrelated to the Function of the Transport System

1998 ◽  
Vol 180 (18) ◽  
pp. 4828-4833 ◽  
Author(s):  
Roberto T. F. Celis ◽  
Peter F. Leadlay ◽  
Ipsita Roy ◽  
Anne Hansen
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Transport System ◽  
Binding Proteins ◽  
Binding Protein ◽  
Transport Systems ◽  
Periplasmic Binding Protein ◽  
Transport Atpase ◽  
Periplasmic Binding Proteins ◽  
K 12

ABSTRACT In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787–1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

1999 ◽  
Vol 82 (09) ◽  
pp. 1177-1181 ◽  
Author(s):  
Hubert de Leeuw ◽  
Pauline Wijers-Koster ◽  
Jan van Mourik ◽  
Jan Voorberg
Keyword(s):  
Endothelial Cells ◽  
Binding Proteins ◽  
Binding Protein ◽  
Control Release ◽  
Small Gtpase ◽  
Gtp Binding Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

1998 ◽  
Vol 79 (04) ◽  
pp. 832-836 ◽  
Author(s):  
Thomas Fischer ◽  
Christina Duffy ◽  
Gilbert White
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

Sign in / Sign up

Export Citation Format

Share Document