A Recombinant Glutamine-Binding Protein from Escherichia coli: Effect of Ligand-Binding on Protein Conformational Dynamics

2004 ◽  
Vol 20 (6) ◽  
pp. 1847-1854 ◽  
Author(s):  
P. Herman ◽  
J. Vecer ◽  
V. Scognamiglio ◽  
M. Staiano ◽  
M. Rossi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Binding Protein ◽  
Conformational Dynamics ◽  
Protein Conformational Dynamics

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.

Pressure effect on the stability and the conformational dynamics of the D-Galactose/D-Glucose-binding protein from Escherichia coli

2005 ◽  
Vol 62 (1) ◽  
pp. 193-201 ◽  
Author(s):  
Anna Marabotti ◽  
Petr Herman ◽  
Maria Staiano ◽  
Antonio Varriale ◽  
Marcella de Champdoré ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pressure Effect ◽  
Binding Protein ◽  
Conformational Dynamics ◽  
Glucose Binding ◽  
The Stability ◽  
Glucose Binding Protein

scholarly journals Ligand binding specificity of the Escherichia coli periplasmic histidine binding protein, HisJ

2016 ◽  
Vol 26 (2) ◽  
pp. 268-279 ◽  
Author(s):  
Subrata Paul ◽  
Sambuddha Banerjee ◽  
Hans J. Vogel
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Binding Protein ◽  
Binding Specificity

scholarly journals Ligand-bound glutamine binding protein assumes multiple metastable binding sites with different binding affinities

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Lu Zhang ◽  
Shaowen Wu ◽  
Yitao Feng ◽  
Dan Wang ◽  
Xilin Jia ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Binding Sites ◽  
Binding Protein ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Affinities ◽  
Binding Mechanism

AbstractProtein dynamics plays key roles in ligand binding. However, the microscopic description of conformational dynamics-coupled ligand binding remains a challenge. In this study, we integrate molecular dynamics simulations, Markov state model (MSM) analysis and experimental methods to characterize the conformational dynamics of ligand-bound glutamine binding protein (GlnBP). We show that ligand-bound GlnBP has high conformational flexibility and additional metastable binding sites, presenting a more complex energy landscape than the scenario in the absence of ligand. The diverse conformations of GlnBP demonstrate different binding affinities and entail complex transition kinetics, implicating a concerted ligand binding mechanism. Single molecule fluorescence resonance energy transfer measurements and mutagenesis experiments are performed to validate our MSM-derived structure ensemble as well as the binding mechanism. Collectively, our study provides deeper insights into the protein dynamics-coupled ligand binding, revealing an intricate regulatory network underlying the apparent binding affinity.

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