ABSTRACT
This paper describes the design, fabrication, and feasibility of paper-based optode devices (PODs) for sensing potassium selectively in biological fluids. PODs operate in exhaustive mode and integrate with a handheld, smartphone-connected optical reader. This integrated measuring system provides significant advantages over traditional optode membranes and other paper-based designs, by obtaining a linear optical response to potassium concentration via a simple, stackable design and by harnessing a smartphone to provide an easy-to-use interface, thus enabling remote monitoring of diseases.
Subject(s)
Potassium , SmartphoneABSTRACT
CheY is a response regulator of bacterial chemotaxis that is activated by phosphorylation of a conserved aspartate residue. However, studies of CheY-phosphate have proven challenging due to rapid hydrolysis of the aspartyl-phosphate in vitro. To combat this issue, we have designed a stable analog suitable for structural and functional studies. Herein, we describe a method for the chemical modification of Thermotoga maritima CheY to produce a phospho-analog designated as phosphono-CheY. Our modification produces a stable analog in the constitutively active form that enables the study of signal transfer to the downstream target.
Subject(s)
Methyl-Accepting Chemotaxis Proteins/chemical synthesis , Thermotoga maritima/metabolism , Bacterial Proteins/chemical synthesis , Bacterial Proteins/chemistry , Chemotaxis , Enzyme Activation , Methyl-Accepting Chemotaxis Proteins/chemistry , Phosphorylation , Protein Stability , Signal Transduction , Thermotoga maritima/chemistryABSTRACT
Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY-phosphate have been challenging due to the rapid hydrolysis of the aspartyl-phosphate in vitro. An inert analog of Tm CheY-phosphate, phosphono-CheY, was synthesized by chemical modification and purified by cation-exchange chromatography. Changes in HPLC retention times, chemical assays for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density for the phosphonomethyl group at Cys54, consistent with a modification at that position. Subsequent biochemical experiments confirmed that protein crystals were phosphono-CheY. Isothermal titration calorimetry and fluorescence polarization binding assays demonstrated that phosphono-CheY bound a peptide derived from FliM, a native partner of CheY-phosphate, with a dissociation constant of â¼29 µM, at least sixfold more tightly than unmodified CheY. Taken together these results suggest that Tm phosphono-CheY is a useful and unique analog of Tm CheY-phosphate.
