Structural origin of weakly ordered nitroxide motion in spin‐labeled proteins

Abstract

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 bsheets. 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 a AHÁÁÁS d 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, Ká lai, 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.

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy

“…This leads to an overestimated flexibility of the SL, which is reflected by broader distance distributions, as previously reported as well (68). By considering the intraresidue C a -H •••S d interaction (48,66,69), the width of the distributions is slightly reduced, but the most probable distances differ as well (Figs. S5, S11, and S13).…”

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

“…1A, Inset) was introduced at solvent-exposed sites to serve as a sensor of local structure. At such sites, R1 typically has weak or no interactions with the protein, as reflected by a singlecomponent electron paramagnetic resonance (EPR) spectrum, and introduces little structural perturbation (16)(17)(18)(19). The R1 sensor Significance Analysis of protein packing reveals the prevalence of cavities, some of which are evolutionarily conserved.…”

“…1A shows a model of the L121A/L133A mutant based on the crystal structure, along with the location of R1 sensor sites and the spectra compared with those of the WT′. The spectra for the WT′ protein have been published previously and interpreted in terms of the protein structure and dynamics (16)(17)(18)(19)(20); the basic principles of spectral analysis are outlined in SI Materials and Methods. The spectra of 131R1, 132R1, 140R1, and 151R1 in the WT′ reflect a simple anisotropic motion characteristic of R1 at solvent-exposed sites in relatively rigid helical segments, whereas the spectra of 48R1, 109R1, 123R1, 128R1, and 135R1 reflect higher mobility of the nitroxide consistent with their location near or at the helix termini (16).…”

“…Taking the hydrophobic free energy to be ≈−25 cal/Å 2 (35) gives ≈−1 kcal of favorable interaction. Based on the current model for the internal motion of R1 at helix surface sites (17,20,36), the adsorption would entail an entropy loss due to restriction of rotamers about the two terminal bonds of R1, each of which is taken to have two most probable configurations (37). This yields an unfavorable contribution to the free energy of −RTln(1/4) ≈ 0.8 kcal/mol, giving a net favorable interaction of ΔG o int ≈ −0.200 kcal/mol.…”

“…Mutation of the solventexposed Y139 to alanine in the W138A background dramatically reduces the component in the 140R1 spectrum that corresponds to the immobile state (Fig. 1B), indicating an important role for Y139 in stabilizing the new conformation; the mutation Y139A has little effect on the WT protein (17). An attractive model for the new conformation is one in which Y139 confers stability by filling the cavity.…”