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Abstract Sinorhizobium melilotisenses nutrients and compounds exuded from alfalfa host roots and coordinates an excitation, termination, and adaptation pathway during chemotaxis. We investigated the role of the novelS. melilotichemotaxis protein CheT. While CheT and theEscherichia coliphosphatase CheZ share little sequence homology, CheT is predicted to possess an α‐helix with a DXXXQ phosphatase motif. Phosphorylation assays demonstrated that CheT dephosphorylates the phosphate‐sink response regulator, CheY1~P by enhancing its decay two‐fold but does not affect the motor response regulator CheY2~P. Isothermal Titration Calorimetry (ITC) experiments revealed that CheT binds to a phosphomimic of CheY1~P with a KDof 2.9 μM, which is 25‐fold stronger than its binding to CheY1. Dissimilar chemotaxis phenotypes of the ΔcheTmutant andcheTDXXXQ phosphatase mutants led to the hypothesis that CheT exerts additional function(s). A screen for potential binding partners of CheT revealed that it forms a complex with the methyltransferase CheR. ITC experiments confirmed CheT/CheR binding with a KDof 19 μM, and a SEC‐MALS analysis determined a 1:1 and 2:1 CheT/CheR complex formation. Although they did not affect each other's enzymatic activity, CheT binding to CheY1~P and CheR may serve as a link between signal termination and sensory adaptation.
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Surface plasmon resonance aptasensing and computational analysis of Staphylococcus aureus IsdA surface protein
Abstract Staphylococcus aureus(S. aureus), a common foodborne pathogen, poses significant public health challenges due to its association with various infectious diseases. A key player in its pathogenicity, which is the IsdA protein, is an essential virulence factor inS. aureusinfections. In this work, we present an integrated in‐silico and experimental approach using MD simulations and surface plasmon resonance (SPR)‐based aptasensing measurements to investigateS. aureusbiorecognition via IsdA surface protein binding. SPR, a powerful real‐time and label‐free technique, was utilized to characterize interaction dynamics between the aptamer and IsdA protein, and MD simulations was used to characterize the stable and dynamic binding regions. By characterizing and optimizing pivotal parameters such as aptamer concentration and buffer conditions, we determined the aptamer's binding performance. Under optimal conditions of pH 7.4 and 150 mM NaCl concentration, the kinetic parameters were determined;ka = 3.789 × 104/Ms,kd = 1.798 × 103/s, andKD = 4.745 × 10−8 M. The simulations revealed regions of interest in the IsdA‐aptamer complex. Region I, which includes interactions between amino acid residues H106 and R107 and nucleotide residues 9G, 10U, 11G and 12U of the aptamer, had the strongest interaction, based on ΔG and B‐factor values, and hence contributed the most to the stability of the interaction. Region II, which covers residue 37A reflects the dynamic nature of the interaction due to frequent contacts. The approach presents a rigorous characterization of aptamer‐IsdA binding behavior, supporting the potential application of the IsdA‐binding aptamer system forS. aureusbiosensing.
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- Award ID(s):
- 2130658
- PAR ID:
- 10570306
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Biotechnology Progress
- Volume:
- 40
- Issue:
- 5
- ISSN:
- 8756-7938
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1002/btpr.3475
