Copper Based Site-directed Spin Labeling of Proteins for Use in Pulsed and Continuous Wave EPR Spectroscopy.

ABSTRACT: Site-directed spin labeling in conjunction with electron paramagnetic resonance (EPR) is an attractive approach to measure residue specific dynamics and point-to-point distance distributions in a biomolecule. Here, we focus on the labeling of proteins with a Cu(II)-nitrilotriacetic acid (NTA) complex, by exploiting two strategically placed histidine residues (called the dHis motif). This labeling strategy has emerged as a means to overcome key limitations of many spin labels. Through utilizing the dHis motif, Cu(II)NTA rigidly binds to a protein without depending on cysteine residues. This protocol outlines three major points: the synthesis of the Cu(II)NTA complex; the measurement of continuous wave and pulsed EPR spectra, to verify a successful synthesis, as well as successful protein la

SUBMITTER: Singewald K 

PROVIDER: S-EPMC8720523 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature