6c40
CheY41PyTyrD54K from Thermotoga maritima
Structural highlights
FunctionCHEY_THEMA Involved in the transmission of sensory signals from the chemoreceptors to the flagellar motors. CheY seems to regulate the clockwise (CW) rotation (By similarity). Publication Abstract from PubMedPulsed dipolar electron spin resonance spectroscopy (PDS) is a powerful tool for measuring distances in solution-state macromolecules. Paramagnetic metal ions, such as Cu(2+), are used as spin probes because they can report on metalloprotein features and can be spectroscopically distinguished from traditional nitroxide (NO)-based labels. Here, we demonstrate site-specific incorporation of Cu(2+) into non-metalloproteins through the use of a genetically encodable non-natural amino acid, 3-pyrazolyltyrosine (PyTyr). We first incorporate PyTyr in cyan fluorescent protein to measure Cu(2+)-to-NO distances and examine the effects of solvent conditions on Cu(2+) binding and protein aggregation. We then apply the method to characterize the complex formed by the histidine kinase CheA and its target response regulator CheY. The X-ray structure of CheY-PyTyr confirms Cu labeling at PyTyr but also reveals a secondary Cu site. Cu(2+)-to-NO and Cu(2+)-to-Cu(2+) PDS measurements of CheY-PyTyr with nitroxide-labeled CheA provide new insights into the conformational landscape of the phosphotransfer complex and have implications for kinase regulation. Site-Specific Incorporation of a Cu(2+) Spin Label into Proteins for Measuring Distances by Pulsed Dipolar Electron Spin Resonance Spectroscopy.,Merz GE, Borbat PP, Muok AR, Srivastava M, Bunck DN, Freed JH, Crane BR J Phys Chem B. 2018 Oct 3. doi: 10.1021/acs.jpcb.8b05619. PMID:30222354[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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