Oxidative stress-mediated protein conformation changes: ESR study of spin-labelled staphylococcal nuclease

We report on the electron spin resonance (ESR) study of the photo-oxidative stress-mediated protein conformation changes in the spin-labelled protein staphylococcal nuclease (SNase). The photo-oxidative stress was brought on by photosensitization of singlet oxygen (1Deltag) in the presence of a novel photosensitizer, water-soluble fullerol C60(OH)19(ONa)17, and resulted in partial protein denaturation. This process was monitored via ESR measurements performed for a spin-labelled SNase Thr-62-Cys mutant, with MTSSL spin label (SL) attached to the cysteine 62 residue (SNase T62C-SL). Prior to ESR measurements of the oxidative stress-induced alterations in protein conformations, the efficiency of C60(OH)19(ONa)17 for 1Deltag-generation was confirmed by three different techniques: (i) selective reactive scavenging of 1Deltag and ESR detection of the resulting paramagnetic product, (ii) 1Deltag-mediated photo-oxidative loss of tryptophan and (iii) by measuring the characteristic near-infrared phosphorescence of singlet oxygen at 1270 nm. The observed evolution of the ESR spectra of SNase T62C-SL as a function of exposure to the photo-oxidative stress points to marked changes in protein conformation due to the deleterious action of 1Deltag.