title: DEER Sensitivity between Iron Centers and Nitroxides in Heme-Containing Proteins Improves Dramatically Using Broadband, High-Field EPR.
abstract: This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron-electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared with previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by taking advantage of recent instrumental advances, employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins.
authors.affiliation: SUPA, School of Physics & Astronomy, University of St Andrews , North Haugh, St. Andrews, Fife, KY16 9SS, United Kingdom.
authors.forename: Claire L
authors.initials: CL
authors.lastname: Motion
authors.affiliation: SUPA, School of Physics & Astronomy, University of St Andrews , North Haugh, St. Andrews, Fife, KY16 9SS, United Kingdom.
authors.forename: Janet E
authors.initials: JE
authors.lastname: Lovett
authors.affiliation: SUPA, School of Physics & Astronomy, University of St Andrews , North Haugh, St. Andrews, Fife, KY16 9SS, United Kingdom.
authors.forename: Stacey
authors.initials: S
authors.lastname: Bell
authors.affiliation: SUPA, School of Physics & Astronomy, University of St Andrews , North Haugh, St. Andrews, Fife, KY16 9SS, United Kingdom.
authors.forename: Scott L
authors.initials: SL
authors.lastname: Cassidy
authors.affiliation: SUPA, School of Physics & Astronomy, University of St Andrews , North Haugh, St. Andrews, Fife, KY16 9SS, United Kingdom.
authors.forename: Paul A S
authors.initials: PA
authors.lastname: Cruickshank
authors.affiliation: SUPA, School of Physics & Astronomy, University of St Andrews , North Haugh, St. Andrews, Fife, KY16 9SS, United Kingdom.
authors.forename: David R
authors.initials: DR
authors.lastname: Bolton
authors.affiliation: SUPA, School of Physics & Astronomy, University of St Andrews , North Haugh, St. Andrews, Fife, KY16 9SS, United Kingdom.
authors.forename: Robert I
authors.initials: RI
authors.lastname: Hunter
authors.affiliation: SUPA, School of Physics & Astronomy, University of St Andrews , North Haugh, St. Andrews, Fife, KY16 9SS, United Kingdom.
authors.forename: Hassane
authors.initials: H
authors.lastname: El Mkami
authors.affiliation: Department of Physics, University of Antwerp , Antwerp 2610, Belgium.
authors.forename: Sabine
authors.initials: S
authors.lastname: Van Doorslaer
authors.affiliation: SUPA, School of Physics & Astronomy, University of St Andrews , North Haugh, St. Andrews, Fife, KY16 9SS, United Kingdom.
authors.forename: Graham M
authors.initials: GM
authors.lastname: Smith
mesh_terms: D000818:Animals; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005914:Globins / Q000737:chemistry*; D006801:Humans; D007501:Iron / Q000737:chemistry*; D008958:Models, Molecular; D009211:Myoglobin / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D000078787:Neuroglobin; D009589:Nitrogen Oxides / Q000737:chemistry*; D011487:Protein Conformation; D013113:Spin Labels
chemical_list: D009211:Myoglobin; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D009589:Nitrogen Oxides; D013113:Spin Labels; D005914:Globins; D007501:Iron; C039900:nitroxyl