title: The double-histidine Cu²⁺-binding motif: a highly rigid, site-specific spin probe for electron spin resonance distance measurements.
abstract: The development of ESR methods that measure long-range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein-backbone structure. Herein we present the double-histidine (dHis) Cu(2+)-binding motif as a rigid spin probe for double electron-electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X-ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 Å of the experimental value. Cu(2+) DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein-backbone structure and flexibility.
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA 15260 (USA).
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Cunningham
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA 15260 (USA).
authors.forename: Miriam R
authors.initials: MR
authors.lastname: Putterman
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA 15260 (USA).
authors.forename: Astha
authors.initials: A
authors.lastname: Desai
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA 15260 (USA). horne@pitt.edu.
authors.forename: W Seth
authors.initials: WS
authors.lastname: Horne
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA 15260 (USA). sksaxena@pitt.edu.
authors.forename: Sunil
authors.initials: S
authors.lastname: Saxena
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006639:Histidine / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D013113:Spin Labels; D013291:Streptococcus / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D013113:Spin Labels; D006639:Histidine; D003300:Copper
keywords: DEER; EPR spectroscopy; copper; proteins; spin labeling
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