CheY is the response regulator protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The chemotaxis (Che) proteins couple flagellar rotation to the environment by transducing chemotactic signals from specific transmembrane chemoreceptors to the flagellar switch apparatus. One of these proteins, CheY, is a small (14-kDa), single-domain protein homologous to the regulator proteins of bacterial two-component sensory transduction systems (3) and is the only two-component signaling protein for which the high-resolution crystal structure has been determined (47, 51). Like other regulators, CheY activity appears to be controlled by phosphorylation (reviewed in references 8 and 48); cheY function is required for CW flagellar rotation (34, 35); CheY is phosphorylated in vitro by CheA (16,17,33,54); and cheY or cheA mutations that disrupt phosphotransfer reactions also result in smoothswimming phenotypes, suggesting that CheY-P is the active CW generator (9,33). While CheY is thought to act directly on the switch apparatus to regulate flagellar rotation (11,38,39,53,55), exactly how CheY functions is not known.

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1H- and 31P-n.m.r. have been used to study the interaction of the bacterial chemotaxis protein, CheY, with ATP and a variety of other phosphates in the presence and absence of bivalent metal ions. In the metal-bound conformation, CheY will bind nucleotide phosphates and phosphates in general, while in the metal-free conformation CheY loses its affinity for phosphates. In the presence of low concentrations of nitroxide-spin-labelled ATP (SL-ATP), specific proton resonances of metal-bound CheY are suppressed, indicating that ATP binds to a specific site on this metal-bound form of the protein. These studies also show that the same resonances are affected by the binding of SL-ATP and Mn2+, indicating that the phosphate- and metal-binding sites are close to each other and to Asp-57 (the site of phosphorylation in CheY). 1H- and 31P-n.m.r. studies using ATP, GTP, TTP, UTP, ADP, AMP and inorganic phosphates show that the binding is not specific for adenine, and does not involve the base directly, but is mediated primarily by the phosphate groups. Experiments with a phosphorylation mutant (Asp-13-->Asn) suggest that the observed phosphate binding and activation of CheY by phosphorylation may be related. Our results indicate that the conformational change and charge interactions brought about by the binding of a metal ion at the active site are required for CheY to interact with a phosphate. These studies also demonstrate the utility of spin-label-induced relaxation in conjunction with two-dimensional-n.m.r. measurements for exploring ligand-binding sites.

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Summary Painful peripheral polyneuropathy is a common complication of diabetes mellitus (DM) and is a significant source of chronic disability and remains a challenging condition with no available disease-modifying treatment. In the present case report, we describe the treatment of a patient featuring painful diabetic neuropathy with perineural injections of autologous plasma rich in growth factors (PRGF). At one-year post-procedure, the patient exhibited improved scores on the neuropathic pain scale and improvement in the activity level. Learning points Plasma rich in growth factors (PRGF) is an autologous product that can be prepared and administered in a physician’s office. PRGF can be infiltrated as a liquid, creating a three-dimensional gel scaffold in the body. PRGF releases growth factors involved in nerve healing. PRGF may be established as a potent alternative treatment of painful diabetic polyneuropathy.

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