Membrane Protein Structure and Dynamics Studied by Site-Directed Spin-Labeling ESR

  • Enrica Bordignon
  • Heinz-Jürgen Steinhoff
Part of the Biological Magnetic Resonance book series (BIMR, volume 27)


ESR spectroscopy of site-directed spin-labeled biomolecules (Site-Directed Spin Labeling, SDSL) has emerged as a powerful method for studying the structure and conformational dynamics of proteins and nucleic acids under conditions relevant to function (for reviews see, e.g., Feix and Klug 1998; Hubbell et al. 1996; Hubbell et al. 1998, 2002). In this technique a spin-label side chain is introduced at a selected site via cysteine substitution mutagenesis followed by modification of the unique sulfhydryl group with a specific paramagnetic nitroxide reagent. The continuous wave (cw) ESR spectrum yields information about the nitroxide side chain mobility, solvent accessibility, the polarity of its immediate environment, and the distance between the nitroxide and another paramagnetic center in the protein. Hence, ESR data analysis of a series of spin-labeled variants of a given protein allows defining elements of secondary structure, including their solvent exposure, to characterize protein topography and to determine orientations of individual segments of the protein. A complete analysis allows modeling of protein structures with a spatial resolution at the level of the backbone fold (Hubbell et al. 1998, 2000; Koteiche and Mchaourab 1999; Mchaourab and Perozo 2000; Perozo et al. 1998; Wegener et al. 2001a). This method is applicable to any protein with a cloned gene that can be expressed. In particular, it has been shown to be very useful in studying large membrane proteins or protein complexes that are not amenable to NMR methods or do not crystallize.


Spin Label Purple Membrane Membrane Protein Structure Interspin Distance Sensory Rhodopsin 
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© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Enrica Bordignon
    • 1
  • Heinz-Jürgen Steinhoff
    • 1
  1. 1.Universität OsnabrückOsnabrückGermany

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