title: Long-range distance measurements in proteins at physiological temperatures using saturation recovery EPR spectroscopy.
abstract: Site-directed spin labeling in combination with EPR is a powerful method for providing distances on the nm scale in biological systems. The most popular strategy, double electron-electron resonance (DEER), is carried out at cryogenic temperatures (50-80 K) to increase the short spin-spin relaxation time (T2) upon which the technique relies. A challenge is to measure long-range distances (20-60 Å) in proteins near physiological temperatures. Toward this goal we are investigating an alternative approach based on the distance-dependent enhancement of spin-lattice relaxation rate (T1(-1)) of a nitroxide spin label by a paramagnetic metal. With a commonly used nitroxide side chain (R1) and Cu(2+), it has been found that interspin distances ≤25 Å can be determined in this way (Jun et al. Biochemistry 2006, 45, 11666). Here, the upper limit of the accessible distance is extended to ≈40 Å using spin labels with long T1, a high-affinity 5-residue Cu(2+) binding loop inserted into the protein sequence, and pulsed saturation recovery to measure relaxation enhancement. Time-domain Cu(2+) electron paramagnetic resonance, quantum mechanical calculations, and molecular dynamics simulations provide information on the structure and geometry of the Cu(2+) loop and indicate that the metal ion is well-localized in the protein. An important aspect of these studies is that both Cu(2+)/nitroxide DEER at cryogenic temperatures and T1 relaxation measurements at room temperature can be carried out on the same sample, allowing both validation of the relaxation method and assessment of the effect of freezing on protein structure.
authors.affiliation: Jules Stein Eye Institute and ‡Department of Chemistry and Biochemistry, University of California , Los Angeles, California 90095, United States.
authors.forename: Zhongyu
authors.initials: Z
authors.lastname: Yang
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Jiménez-Osés
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: López
authors.forename: Michael D
authors.initials: MD
authors.lastname: Bridges
authors.forename: K N
authors.initials: KN
authors.lastname: Houk
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
mesh_terms: D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology; D001665:Binding Sites; D003300:Copper / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009589:Nitrogen Oxides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011789:Quantum Theory; D012399:Rotation; D013113:Spin Labels; D013696:Temperature
chemical_list: D009589:Nitrogen Oxides; D010455:Peptides; D011506:Proteins; D013113:Spin Labels; D003300:Copper; D009113:Muramidase; C039900:nitroxyl