Dataset Information

Identification and removal of nitroxide spin label contaminant: impact on PRE studies of ?-helical membrane proteins in detergent.

ABSTRACT: NMR paramagnetic relaxation enhancement (PRE) provides long-range distance constraints (~15-25 Å) that can be critical to determining overall protein topology, especially where long-range NOE information is unavailable such as in the case of larger proteins that require deuteration. However, several challenges currently limit the use of NMR PRE for ?-helical membrane proteins. One challenge is the nonspecific association of the nitroxide spin label to the protein-detergent complex that can result in spurious PRE derived distance restraints. The effect of the nitroxide spin label contaminant is evaluated and quantified and a robust method for the removal of the contaminant is provided to advance the application of PRE restraints to membrane protein NMR structure determination.

SUBMITTER: Kroncke BM

PROVIDER: S-EPMC3375759 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Identification and removal of nitroxide spin label contaminant: impact on PRE studies of α-helical membrane proteins in detergent.

Kroncke Brett M BM Columbus Linda L

Protein science : a publication of the Protein Society 20120302 4

NMR paramagnetic relaxation enhancement (PRE) provides long-range distance constraints (~15-25 Å) that can be critical to determining overall protein topology, especially where long-range NOE information is unavailable such as in the case of larger proteins that require deuteration. However, several challenges currently limit the use of NMR PRE for α-helical membrane proteins. One challenge is the nonspecific association of the nitroxide spin label to the protein-detergent complex that can resul ...[more]

PMID: 22389096

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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

Identification and removal of nitroxide spin label contaminant: impact on PRE studies of ?-helical membrane proteins in detergent.

Publications

Identification and removal of nitroxide spin label contaminant: impact on PRE studies of α-helical membrane proteins in detergent.

Similar Datasets

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