Structural characterization of the Cu(II)-NTA spin label on α-helices by X-ray crystallography and electron
Jessica E Besaw1, Jörg Reichenwallner1, Evelyn Y Chen1
1Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
Abstract:
Site-directed Cu(II)-labelling in pulsed electron paramagnetic resonance (EPR) spectroscopy has demonstrated narrow Cu(II)-Cu(II) distance distributions suitable to resolve subtle protein conformational changes. The high precision derives from a double histidine (dHis) mutation that effectively locks a Cu(II)-nitrilotriacetic acid (Cu(II)-NTA) moiety in place. To date, no structures featuring the dHis-Cu(II)-NTA motif have been resolved. This work presents the atomic-resolution X-ray crystal structures of seven α-helical dHis sites of T4 lysozyme (T4L) in the presence and absence of Cu(II)-NTA. Our research captured the rigid octahedral coordination of the dHis-Cu(II)-NTA complex as well as non-conventional binding modes, which provide valuable insight into dHis site selection. Pulsed EPR experiments on double dHis T4L mutants displayed remarkable agreement to the crystallography-derived distances. This research showcases the rigid configuration of the dHis-Cu(II)-NTA motif, providing geometric constraints that can be leveraged in modeling and molecular dynamics programs to extract protein structural details from EPR experiments.
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