RESUMEN
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.
Asunto(s)
Sitios de Unión , Ciclohexanoles/química , Diseño de Fármacos , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Modelos Moleculares , Proteínas de Unión Periplasmáticas/química , Aminoácidos , Evaluación Preclínica de Medicamentos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Biblioteca de Genes , Ligandos , Mutación , Proteínas de Unión Periplasmáticas/antagonistas & inhibidores , Proteínas de Unión Periplasmáticas/genética , Relación Estructura-ActividadRESUMEN
Deficiencies in thiamine (vitamin B1) cause a host of neurological and reproductive impairments yielding morbidity and mortality across environmental and clinical realms. In a technique analogous to immunomagnetic separation, we introduce the use of thiamine periplasmic binding protein (TBP)-conjugated magnetic beads to isolate thiamine from complex matrices. TBP expressed in Escherichia coli is highly specific to thiamine and provides an alternative to antibodies for this non-immunogenic target. After incubation with the sample and removal of unbound matrix constituents, thiamine is simultaneously released and converted to its fluorescent oxidation product thiochrome by alkaline potassium ferricyanide. Subsequent measurement of fluorescence at thiochrome-specific wavelengths provides a second layer of specificity for the detection of thiamine. Thiamine could be quantified at concentrations as low as 5â¯nM ranging up to 240â¯nM. Within, we apply this technique to selectively capture and quantify thiamine in complex salmonid fish egg and tissue matrices. Our results showed no measurable non-specific binding to the beads by endogenous fluorophores in the fish egg matrix. Thiamine levels as low as 0.2â¯nmol/g of fish egg can be detected using this approach, which is sufficient to assess deficiencies causing morbidity and mortality in fish that occur at 1.0â¯nmol/g of egg. This practical method may find application in other resource limited settings for clinical, food, or dietary supplement analyses.
Asunto(s)
Técnicas Biosensibles/métodos , Imanes/química , Proteínas de Unión Periplasmáticas/química , Tiamina/análisis , Tiamina/aislamiento & purificación , Transferasas Alquil y Aril/metabolismo , Animales , Huevos/análisis , Límite de Detección , Microesferas , Salmón , Tiamina/metabolismoRESUMEN
In conventional "Venus Flytrap" mechanism, substrate-binding proteins (SBPs) interconvert between the open and closed conformations. Upon ligand binding, SBPs form a tightly closed conformation with the ligand bound at the interface of two domains. This mechanism was later challenged by many type III SBPs, such as the vitamin B12 -binding protein BtuF, in which the apo- and holo-state proteins adopt very similar conformations. Here, we combined molecular dynamics simulation and Markov state model analysis to study the conformational dynamics of apo- and B12 -bound BtuF. The results indicate that the crystal structures represent the only stable conformation of BtuF. Meanwhile, both apo- and holo-BtuF undergo large-scale interdomain motions with little energy cost. B12 binding casts little restraints on the interdomain motions, suggesting that ligand binding affinity is enhanced by the remaining conformational entropy of holo-BtuF. These results reveal a new paradigm of ligand recognition mechanism of SBPs. © 2019 Wiley Periodicals, Inc.
Asunto(s)
Proteínas de Escherichia coli/química , Cadenas de Markov , Simulación de Dinámica Molecular , Proteínas de Unión Periplasmáticas/química , Sitios de Unión , Cristalografía por Rayos X , Cinética , Ligandos , Conformación ProteicaRESUMEN
Gram-negative bacteria use siderophores, outer membrane receptors, inner membrane transporters and substrate-binding proteins (SBPs) to transport transition metals through the periplasm. The SBPs share a similar protein fold that has undergone significant structural evolution to communicate with a variety of differentially regulated transporters in the cell. In Yersinia pestis, the causative agent of plague, YfeA (YPO2439, y1897), an SBP, is important for full virulence during mammalian infection. To better understand the role of YfeA in infection, crystal structures were determined under several environmental conditions with respect to transition-metal levels. Energy-dispersive X-ray spectroscopy and anomalous X-ray scattering data show that YfeA is polyspecific and can alter its substrate specificity. In minimal-media experiments, YfeA crystals grown after iron supplementation showed a threefold increase in iron fluorescence emission over the iron fluorescence emission from YfeA crystals grown from nutrient-rich conditions, and YfeA crystals grown after manganese supplementation during overexpression showed a fivefold increase in manganese fluorescence emission over the manganese fluorescence emission from YfeA crystals grown from nutrient-rich conditions. In all experiments, the YfeA crystals produced the strongest fluorescence emission from zinc and could not be manipulated otherwise. Additionally, this report documents the discovery of a novel surface metal-binding site that prefers to chelate zinc but can also bind manganese. Flexibility across YfeA crystal forms in three loops and a helix near the buried metal-binding site suggest that a structural rearrangement is required for metal loading and unloading.
Asunto(s)
Metales/metabolismo , Proteínas de Unión Periplasmáticas/química , Peste/microbiología , Factores de Virulencia/química , Yersinia pestis/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Humanos , Hierro/metabolismo , Manganeso/metabolismo , Modelos Moleculares , Proteínas de Unión Periplasmáticas/metabolismo , Conformación Proteica , Alineación de Secuencia , Especificidad por Sustrato , Factores de Virulencia/metabolismo , Yersinia pestis/metabolismo , Zinc/metabolismoRESUMEN
Protein function, structure, and dynamics are intricately correlated, but studies on structure-activity relationships are still only rarely complemented by a detailed analysis of dynamics related to function (functional dynamics). Here, we have applied NMR to investigate the functional dynamics in two homologous periplasmic sugar binding proteins with bidomain composition: Escherichia coli glucose/galactose (GGBP) and ribose (RBP) binding proteins. In contrast to their structural and functional similarity, we observe a remarkable difference in functional dynamics: For RBP, the absence of segmental motions allows only for isolated structural adaptations upon carbohydrate binding in line with an induced fit mechanism; on the other hand, GGBP shows extensive segmental mobility in both apo and holo states, enabling selection of the most favorable conformation upon carbohydrate binding in line with a population shift mechanism. Collective segmental motions are controlled by the hinge composition: by swapping two identified key residues between RBP and GGBP we also interchange their segmental hinge mobility, and the doubly mutated GGBP* no longer experiences changes in conformational entropy upon ligand binding while the complementary RBP* shows the segmental dynamics observed in wild-type GGBP. Most importantly, the segmental interdomain dynamics always increase the apparent substrate affinity and thus, are functional, underscoring the allosteric control that the hinge region exerts on ligand binding.
Asunto(s)
Proteínas de Unión al Calcio/química , Glucosa/química , Proteínas de Transporte de Monosacáridos/química , Proteínas de Unión Periplasmáticas/química , Regulación Alostérica , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/metabolismo , Calorimetría , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glucosa/metabolismo , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Unión Periplasmáticas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Recent improvements in wheat-embryo cell-free translation resulted in a highly productive system for protein preparation. To clarify N-terminal processing of the cell-free system in a preparative-scale (> mg protein product per ml), 20 mutant variants of maltose-binding protein (MalE), each having a different penultimate residue in the sequence Met-Xaa-Ile-Glu-, and 20 glutathione S-transferase (GST) variants, having Met-Xaa-Pro-Ile-sequence, were designed and synthesized. The MalE and GST proteins were purified by amylose-resin and glutathione columns, respectively, followed by analysis of their N-terminal sequences. These investigations revealed that sequence specificity and efficiency of the N-terminal Met (N-Met) elimination in the cell-free system are similar to those reported from investigations in cellular systems or in the wheat-embryo cell-free protein expression system in analytical scale (approximately 10 microg protein product per ml). Cleavage of the N-Met is basically determined by the penultimate amino acid in the polypeptide sequence. In the case of MalE, the cleavage was efficient when the penultimate residue was Ala, Cys, Gly, Pro, Ser or Thr. But, in the case of GST with Pro as the antepenultimate residue, the efficiency was significantly reduced when the penultimate residue was Gly or Thr. We also confirmed that substitution of the antepenultimate residue in MalE to Pro drastically reduced the efficiency of N-Met cleavage when the penultimate residue was Ala, Gly, Pro, Ser or Thr, indicating inhibitory effects of antepenultimate residue Pro on N-Met elimination. These results clarified sequence-specific functions of the endogenous N-terminal processing machinery in the scaled-up wheat-embryo cell-free translation system.
Asunto(s)
Metionina/metabolismo , Proteínas de Plantas/metabolismo , Semillas/fisiología , Triticum/fisiología , Secuencia de Aminoácidos , Sistema Libre de Células , Cartilla de ADN , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Proteínas de Unión Periplasmáticas/química , Proteínas de Unión Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/metabolismo , Extractos Vegetales/metabolismo , Proteínas de Plantas/química , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
It is difficult to imagine any strategy for high-throughput protein expression and purification that does not involve genetically engineered affinity tags. Because of its ability to enhance the solubility and promote the proper folding of its fusion partners, Escherichia coli maltose-binding protein (MBP) is a particularly useful affinity tag. However, not all MBP fusion proteins bind efficiently to amylose resin, and even when they do it is usually not possible to obtain a sample of adequate purity after a single affinity step. To address this problem, we endeavored to incorporate supplemental affinity tags within the framework of an MBP fusion protein. We show that both the nature of the supplemental tags and their location can influence the ability of MBP to promote the solubility of its fusion partners. The most promising configurations for high-throughput protein expression and purification appear to be a fusion protein with a biotin acceptor peptide (BAP) on the N-terminus of MBP and/or a hexahistidine tag (His-tag) on the C-terminus of the passenger protein.