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Conformational analysis of a nitroxide side chain in an ?-helix with density functional theory.

ABSTRACT: 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 R1. The electron paramagnetic resonance (EPR) spectra of R1, and the interspin distance between pairs of R1 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 R1 side chain, that is, the favored rotamers, the intraresidue interactions that stabilize them, and the internal modes of motion. X-ray crystal structures of R1 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 R1 in a 10-residue ?-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.

SUBMITTER: Warshaviak DT

PROVIDER: S-EPMC3267783 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Conformational analysis of a nitroxide side chain in an α-helix with density functional theory.

Warshaviak Dora Toledo DT Serbulea Laura L Houk K N KN Hubbell Wayne L WL

The journal of physical chemistry. B 20101217 2

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 R1. The electron paramagnetic resonance (EPR) spectra of R1, and the interspin distance between pairs of R1 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 R1 side chain, t ...[more]

PMID: 21162593

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Project description:Understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. EPR spectroscopy in combination with site-directed spin labeling (SDSL) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin label's intramolecular motion in the hydrophobic environment. Although several studies have elucidated the structural origins of EPR line shapes of water-soluble proteins, EPR spectra of nitroxide spin-labeled proteins in detergents or lipids have characteristic differences from their water-soluble counterparts, suggesting significant differences in the underlying molecular motion of the spin label between the two environments. To elucidate these differences, membrane-exposed ?-helical sites of the leucine transporter, LeuT, from Aquifex aeolicus, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure, the nitroxide ring of a disulfide-linked spin label side chain (R1) is resolved and makes contacts with hydrophobic residues on the protein surface. The spin label at site I204 on LeuT makes a nontraditional hydrogen bond with the ortho-hydrogen on its nearest neighbor F208, whereas the spin label at site F177 makes multiple van der Waals contacts with a hydrophobic pocket formed with an adjacent helix. These results coupled with the spectral effect of mutating the i ± 3, 4 residues suggest that the spin label has a greater affinity for its local protein environment in the low dielectric than on a water-soluble protein surface. The simulations of the EPR spectra presented here suggest the spin label oscillates about the terminal bond nearest the ring while maintaining weak contact with the protein surface. Combined, the results provide a starting point for determining a motional model for R1 on membrane proteins, allowing quantification of nitroxide dynamics in the aliphatic environment of detergent and lipids. In addition, initial contributions to a rotamer library of R1 on membrane proteins are provided, which will assist in reliably modeling the R1 conformational space for pulsed dipolar EPR and NMR paramagnetic relaxation enhancement distance determination.

Dataset Information

Conformational analysis of a nitroxide side chain in an ?-helix with density functional theory.

Publications

Conformational analysis of a nitroxide side chain in an α-helix with density functional theory.

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