Conformational Analysis of a Nitroxide Side Chain in an α-Helix with Density Functional Theory

In site directed spin labeling, a nitroxide side chain is introduced at a selected site in a protein; the most commonly used is a disulfide-linked side chain designated RI. The electron paramagnetic resonance (EPR) spectra of RI, and the interspin distance between pairs of 121 residues as determined by dipolar EPR spectroscopy, encode a wealth of information on the protein structure and dynamics. However, extracting this information requires structural and dynamical models of the 121 side chain, that is, the favored rotamers, the intraresidue interactions that stabilize them, and the internal modes of motion. X-ray crystal structures of 121 in proteins have revealed a set of preferred rotamers in the crystal lattice. To identify the intraresidue interactions that stabilize particular rotamers of R1 in the absence of interactions with nearby side chains in a helix, and to evaluate models for the internal motion of the side chain, quantum mechanical calculations were performed on a relevant fragment of RI in a 10-residue alpha-helix. Relative rotamer energies were determined in the gas phase, and solvation energies were estimated from a continuum solvent model that includes both electrostatic and hydrophobic contributions. The results identified preferred rotamers that are in agreement with the X-ray crystallographic studies. The rotamers are apparently stabilized by intraresidue sulfur-backbone interactions, suggesting that the preferred rotamers may be the same at all solvent-exposed helix sites.