Work

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

Year: 2018

Type: article

Abstract: Pulsed 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.

Source: The Journal of Physical Chemistry B

DOI: 10.1021/acs.jpcb.8b05619