Site-Specific Incorporation of a Cu Spin-Label Into Proteins for Measuring Distances by Pulsed Dipolar ESR Spectroscopy.
Publication Type:Journal Article
Source:J Phys Chem B (2018)
<p>Pulsed dipolar ESR spectroscopy (PDS) is a powerful tool for measuring distances in solution-state macromolecules. Paramagnetic metal ions, such as Cu2+, are useful spin probes because they can report on metalloproteins features and can be spectroscopically distinguished from traditional nitroxide (NO)-based labels. Here we demonstrate site-specific incorporation of Cu2+ 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 Cu2+-to-NO distances and examine the effects of solvent conditions on Cu2+ 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. Cu2+-to-NO and Cu2+-to-Cu2+ 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.</p>