Probing iso-1-cytochrome c structure by site-directed spin labeling and electron paramagnetic resonance techniques.

Abstract

A cysteine-specific methanethiosulfonate spin label was introduced into yeast iso-1-cytochrome c at three different positions. The modified forms of cytochrome c included: the wild-type protein labeled at naturally occurring C102, and two mutated proteins, S47C and L85C, labeled at positions 47 and 85, respectively (both S47C and L85C derived from the protein in which C102 had been replaced by threonine). All three spin-labeled protein derivatives were characterized using electron paramagnetic resonance (EPR) techniques. The continuous wave (CW) EPR spectrum of spin label attached to L85C differed from those recorded for spin label attached to C102 or S47C, indicating that spin label at position 85 was more immobilized and exhibited more complex tumbling than spin label at two other positions. The temperature dependence of the CW EPR spectra and CW EPR power saturation revealed further differences of spin-labeled L85C. The results were discussed in terms of application of the site-directed spin labeling technique in probing the local dynamic structure of iso-1-cytochrome c.

Mapping Local Protein Electrostatics by EPR of pH-Sensitive Thiol-Specific Nitroxide

“…For all pH values, the label tethered at C102, the penultimate C-terminal amino acid residue located in the loop region, experienced only small rotational restrictions as indicated by EPR spectra characteristic for the fast motion regime. This is typical for surface-exposed protein side chains . One could also expect that such a progressive lowering of pH would trigger denaturing of this protein.…”