Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy

Abstract

A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i + 3 or i and i + 4 in an α-helix, at i and i + 2 in a β-strand, or with cysteine residues in adjacent strands in a β-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.

Fig. 1.

The RX side chain, sites of introduction in T4 Lysozyme (T4L) and intestinal fatty acid-binding protein (iFABP), and the corresponding EPR spectra. (A) Reaction of a protein containing two cysteines with the homobifunctional, reagent (HO-1944) results in the cross-linked side chain designated “RX.” (B and C) Ribbon models of wild-type T4L [B, PDB ID code 1L63 (26)] and iFABP [C, PDB ID code 2IFB (27)] highlighting representative solvent-exposed sites used in this study with spheres at their α-carbons. (D and E) Normalized EPR spectra of representative RX mutants of T4L (D) and iFABP (E) attached to CNBr-activated Sepharose in buffer. Overlaid on the Sepharose-bound spectrum for T4L 5-9RX is nonlinear least-squares fit to a MOMD model (red-dashed trace). A vertical scaling factor (Left) was applied to the spectrum of 5-9RX for display purposes. The magnetic field scan width is 100 G. The effective hyperfine splitting () and the parallel (A_||) and perpendicular (A_⊥) components of the axially symmetric hyperfine tensor for the 5-9RX spectrum are shown.

Fig. 2.

Crystal structure of T4L 115-119RX. Electron density for T4L 115-119RX calculated as an unweighted 2F_o - F_c composite omit map (blue mesh) and contoured at 1.0 σ. For clarity, only a stick model and electron density for RX side chain and selected nearby residues are shown with the protein backbone displayed as a ribbon model. Dihedral angles of the RX side chain from 115: {X₁ = -94^°,X₂ = -61°,X₃ = -81°,X₄ = -160°,X₅ = 105°} from 119 : {X₁ = -70°,X₂ = -56°,X₃ = 106°,X₄ = 122°,X₅ = -90°} using the convention in Fig. 1A.

Fig. 3.

DEER data of T4L mutants bearing either two RX side chains (black traces) or two R1 side chains (red traces). Background-subtracted dipolar evolutions of the indicated mutants (Left) and their corresponding area-normalized distance probability distributions from Tikhonov regularization (Right) for (A) T4L 109R1/131R1 and T4L 109-113RX/127-131RX and (B) iFABP 48R1/116R1 and iFABP 48-50RX/114-116RX. The width of the interspin distance distribution (between 16 and 84% probability) is 6.5 Å for T4L 109R1/131R1, 2.8 Å for T4L 109-113RX/127-131RX, 4.7 Å for iFABP 48R1/116R1, and 2.0 Å for iFABP 48-50RX/114-116RX.

Fig. 4.

ELDOR studies of T4L mutants. (A) EPR absorption lineshape for an immobilized nitroxide showing the fields (and nitroxide 2p-orbital orientations, θ) at which the saturating pulse was applied (pump) and at which the arrival and recovery of saturation were observed (observe 1 or observe 2); (B) Intra- (red) and intermanifold (blue) ELDOR curves and global fits to the spectra using both SR and ELDOR data (gray; SI Materials and Methods) for 5–9RX (Upper) and 44-48RX/T26E (Lower) attached to Sepharose in buffer. (C) Intra- (red) and intermanifold (blue) ELDOR curves and global fits to the spectra (gray) of the T4L 5-9RX mutant in buffer (Upper) and in 80% glycerol (Lower) at 298 K. The time constants yielded by the fits in B and C are as follows (T_1e, T_1n, τ_R, respectively, in μs): 5–9RX on Sepharose; 7.79 ± 0.02, 0.91 ± 0.02, not applicable (N/A); 44-48RX/T26E on Sepharose; 7.90 ± 0.04, 0.56 ± 0.09, 6.2 ± 0.3; 5–9RX in buffer; 7.12 ± 0.01, N/A, N/A; 5–9RX in 80% glycerol; 11.1 ± 0.1, 0.22 ± 0.07, 1.2 ± 0.6. Each curve spans 40 μs.