Structural origin of weakly ordered nitroxide motion in spin-labeled proteins.
A disulfide-linked nitroxide side chain (R1) used in site-directed spin labeling of proteins often exhibits an EPR spectrum characteristic of a weakly ordered z-axis anisotropic motion at topographically diverse surface sites, including those on helices, loops and edge strands of beta-sheets. To elucidate the origin of this motion, the first crystal structures of R1 that display simple z-axis anisotropic motion at solvent-exposed helical sites (131 and 151) and a loop site (82) in T4 lysozyme have been determined. Structures of 131R1 and 151R1 determined at cryogenic or ambient temperature reveal an intraresidue C(alpha)--H...S(delta) interaction that immobilizes the disulfide group, consistent with a model in which the internal motions of R1 are dominated by rotations about the two terminal bonds (Columbus, Kálai, Jeko, Hideg, and Hubbell, Biochemistry 2001;40:3828-3846). Remarkably, the 131R1 side chain populates two rotamers equally, but the EPR spectrum reflects a single dominant dynamic population, showing that the two rotamers have similar internal motion determined by the common disulfide-backbone interaction. The anisotropic motion for loop residue 82R1 is also accounted for by a common disulfide-backbone interaction, showing that the interaction does not require a specific secondary structure. If the above observations prove to be general, then significant variations in order and rate for R1 at noninteracting solvent-exposed helical and loop sites can be assigned to backbone motion because the internal motion is essentially constant.
Fleissner
Mark R
MR
Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-7008.
Cascio
Duilio
D
Hubbell
Wayne L
WL
D017122:Bacteriophage T4 / Q000201:enzymology; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry; D009589:Nitrogen Oxides / Q000737:chemistry*; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011506:Proteins / Q000737:chemistry*; D013113:Spin Labels; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry
D004220:Disulfides; D009589:Nitrogen Oxides; D011506:Proteins; D013113:Spin Labels; D014764:Viral Proteins; D009113:Muramidase