Site-Specific Incorporation of a Cu2+ Spin-Label into Proteins for Measuring Distances by Pulsed Dipolar ESR Spectroscopy

Pulsed dipolar ESR spectroscopy (PDS) is a powerful tool for measuring distances in solution-state macromolecules. Paramagnetic metal ions, such as Cu 2+ , are useful spin probes because they can report on metalloproteins features and can be spectroscopically distinguished from traditional ni-troxide (NO)-based labels. Here we demonstrate site-specific incorporation of Cu 2+ into non-metalloproteins through use of a genetically encodable non-natural amino acid, 3-pyrazolyltyrosine (PyTyr). We first incorporate PyTyr in cyan-fluorescent protein (CFP) 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 reg-ulator 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 insight into the conformational landscape of the phosphotransfer complex and have implications for kinase regulation.