6C40

CheY41PyTyrD54K from Thermotoga maritima

Summary for 6C40

• Entry DOI: 10.2210/pdb6c40/pdb
• Descriptor: Chemotaxis protein CheY, COPPER (II) ION (3 entities in total)
• Functional Keywords: chey, chemotaxis, pytyr, signaling protein
• Biological source: Thermotoga maritima MSB8
• Total number of polymer chains: 2
• Total formula weight: 26558.46

Authors

Merz, G.E.,Muok, A.R.,Crane, B.R. (deposition date: 2018-01-11, release date: 2018-10-17, Last modification date: 2023-11-15)

Primary citation

Merz, G.E.,Borbat, P.P.,Muok, A.R.,Srivastava, M.,Bunck, D.N.,Freed, J.H.,Crane, B.R.
Site-Specific Incorporation of a Cu2+Spin Label into Proteins for Measuring Distances by Pulsed Dipolar Electron Spin Resonance Spectroscopy.
J Phys Chem B, 122:9443-9451, 2018

PubMed 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, 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 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-to-NO distances and examine the effects of solvent conditions on Cu 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-to-NO and Cu-to-Cu 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.

PubMed: 30222354
DOI: 10.1021/acs.jpcb.8b05619

Experimental method

X-RAY DIFFRACTION (2.7 Å)