An imidoester spin probe of membrane protein interactions: Application to cytochrome c

Abstract

The synthesis of an imidoester spin label, whose advantages relative to other spin labels include its water solubility, lysine specificity, and retention of positive charge at the reaction site is described. Cytochrome c is spin labeled and shown to exhibit spectral changes upon interacting with lipid vesicles and lipid-rich cytochrome oxidase preparations. Spin labeled cytochrome c in buffer or in the presence of mitochondria at high ionic strength had a correlation time of τ = 0.91 ± 10 −9 s; at low ionic strength the mitochondrial signal was more immobilized, τ = 2.27 ± 0.13 × 10 −9 s; and further immobilization was observed when cytochrome c was bound to the high-affinity site of purified oxidase containing 37% phospholipid (τ = 2.71 ± 0.22 × 10 −9). Cytochrome c-oxidase electron transfer rates were unaltered by spin labeling. The results suggest that this imidoester spin label will be useful for studies of protein-protein and protein-lipid interactions.