Abstact

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 alpha-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.