6c40

Publication Abstract from PubMed

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.

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.