Structure of the nitroxide side chain R1 showing designation of the various atoms and side-chain dihedral angles (χ). χ₅ is defined by S_δ−C_ε−C₃−C₄ as in Tombolato et al. (2006).

Structure of 41R1. (A) Electron density superimposed on a stick model of 41R1. The 2F _o−F _c map is contoured at 1.3σ. (B) Distance of the S_δ atom of the disulfide group from the H atom modeled on 41C_α. (C) Distances of atoms in the nitroxide ring from nearest neighbors in the lattice. A symmetry-related molecule is colored cyan, and residues belonging to this molecule are indicated with a prime (′). (D) Space-filling model of 41R1 and neighboring groups.

Structures of 44R1. (A) Electron density superimposed on a stick model of 44R1 corresponding to the A, B, and C chains in the lattice; residue E45 is also shown. The 2F _o−F _c maps are contoured at 1.3σ, 1.2σ, and 1.1σ for 44R1_A, 44R1_B, and 44R1_C, respectively. (B) Distances from the S_δ of the disulfide to the H atom modeled on 44C_α are shown. Contacts of the nitroxide ring of 44R1_A and 44R1_B with symmetry-related molecules are too numerous to show clearly.

Analysis of the EPR spectra of 44R1 and 80R1 with an X-axis anisotropy model. (A−C) EPR spectra of 44R1, 44R1/E45A, and 80R1 (solid traces) together with fits based on X-axis anisotropy for the mobile component (dashed traces). Individual components derived from the fits are shown to the right. For 44R1, i and m components for the fit have τ = 13 ns (isotropic) and τ = 1.9 ns, S = 0.44, β_D = 90, γ_D = 36, respectively. For 44R1/E45A, i and m components for the fit have τ = 13 ns (isotropic) and τ = 2.0 ns, S = 0.34, β_D = 90, γ_D = 36, respectively. The fit for 80 R1 has τ = 1.5 ns, S = 0.37, β_D = 90, γ_D = 35. (Inset) Diagram showing the relationship between the nitroxide molecular frame (x _M, y _M, and z _M) and the z-axis of the diffusion tensor (z _R) in X-axis anisotropy. For simplicity, the methyl substituents at positions 2 and 5 of the nitroxide ring are not shown.

Possible intrahelical interactions of R1. (A) Left panel: R1 in the {m,m} rotamer of the 44R1_A crystal structure (Table 3). Right panel: 44R1_A in a conformation in which χ₃ has isomerized from −95° to 90°. (B) Left panel: R1 in the {t,p} rotamer of the 41R1 crystal structure (Table 3). The nitroxide ring was modeled using χ₄ = −75° and χ₅ = 100°. Right panel: 41R1 in a conformation in which χ₃ is isomerized from 86° to −96° and χ₄ and χ₅ are −80° and 0°, respectively. (C) R1 in a {t,m} rotamer. Dihedral angles χ₃, χ₄, and χ₅ are modeled as −90°, 180°, and 28°, respectively.

Sites in T4L where R1 was placed along helix B and the corresponding EPR spectra. (A) Ribbon diagram of the T4L structure. Spheres at the C_α atoms identify residues where R1 has been introduced. The C_α atoms of the most solvent-exposed sites (fractional side-chain solvent accessibility >0.4) are shown in magenta. (B) EPR spectra of R1 at the indicated positions. Arrows “i” and “m” indicate spectral components that arise from immobile and mobile states of R1, respectively. Resolved hyperfine extrema (2A_zz′) are identified for 50R1.

Effect of mutations of neighbor residues on the EPR spectra of 41R1 in solution. (A−E) EPR spectra of 41R1 in the context of indicated mutations (solid traces) and fits to 41R1 and 41R1/E45A (dashed traces). The individual components determined from the fits are shown to the right. For 41R1, i and m components for the fit have τ = 10.5 ns (isotropic) and τ = 1.5 ns, S = 0.37, and β_D = 36, respectively. For 41R1/E45A, i and m components for the fit have τ = 10.5 ns (isotropic) and τ = 1.6 ns, S = 0.31, and β_D = 36, respectively. Arrowheads on the mobile component of 41R1 indicate resolved parallel and perpendicular orientations with respect to the magnetic field. (Inset) A diagram showing the relationship between the nitroxide molecular frame (x _M, y _M, and z _M) and the z-axis of the diffusion tensor (z _R) for z-axis anisotropic motion. For simplicity, the methyl substituents at positions 2 and 5 of the nitroxide ring are not shown. As is customary, z _M lies along the nitroxide p orbital, x _M lies along the N−O bond, and y _M is selected for a right-handed coordinate system.

Effect of mutations of neighbor residues on the EPR spectra of 44R1 in solution. (A) Ribbon diagram of T4L showing the position of 44 (sphere) and neighboring residues (stick models). (B) EPR spectra of 44R1 in the context of indicated mutations. For comparison, the 44R1 spectrum (gray trace) is superimposed on all other spectra.

Rotamer preferences of R1 at solvent-exposed helical sites. Side-chain dihedral angles χ₁ and χ₂ from the present work are shown by circles, and those from Langen et al. (2000) and Guo et al. (2007) are shown by triangles. Dashed boxes identify ±30° about the average values for the dihedral angles.