Structure and dynamics of an imidazoline nitroxide side chain with strongly hindered internal motion in proteins

Abstract

A disulfide-linked imidazoline nitroxide side chain (V1) has a similar and highly constrained internal motion at diverse topological sites in a protein, unlike that for the disulfide-linked pyrroline nitroxide side chain (R1) widely used in site directed spin labeling EPR. Crystal structures of V1 at two positions in a helix of T4 Lysozyme and quantum mechanical calculations suggest the source of the constraints as intra-side chain interactions of the disulfide sulfur atoms with both the protein backbone and the 3-nitrogen in the imidazoline ring. These interactions apparently limit the conformation of the side chain to one of only three possible rotamers, two of which are observed in the crystal structure. An inter-spin distance measurement in frozen solution using double electron-electron resonance (DEER) gives a value essentially identical to that determined from the crystal structure of the protein containing two copies of V1, indicating that lattice forces do not dictate the rotamers observed. Collectively, the results suggest the possibility of predetermining a unique rotamer of V1 in helical structures. In general, the reduced rotameric space of V1 compared to R1 should simplify interpretation of inter-spin distance information in terms of protein structure, while the highly constrained internal motion is expected to extend the dynamic range for characterizing large amplitude nanosecond backbone fluctuations.

Figure 1

Introduction of nitroxide side chains via cysteine substitution mutagenesis. In each case, a cysteine residue is introduced at the site of interest, followed by reaction with the desired sulfhydryl specific reagent. (A), reaction with 1-Oxyl-2,2,5,5-tetramethypyrroline-3-methyl (MTSL) to generate R1. (B), reaction with bis(2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-il)-disulfide (IDSL) to generate V1. Numbering of dihedral angles (X) is shown for each side chain.

Figure 2

The EPR spectrum of T4L 72V1 in 30% sucrose solution. (A) The EPR spectrum was obtained immediately following reaction with IDSL and separation of excess reagent. (B) The EPR spectrum obtained after cleaving the IDSL disulfide bond with equimolar DTT. (C) The result of subtracting the spectrum in (B) from that in (A); the sharp component in this case amounts to ≈ 2.5% of the total spin concentration.

Figure 3

Comparison of EPR spectra of R1 and V1 at sites in T4L. (A) A ribbon Model of T4L, showing the investigated sites. (B) Room temperature spectra of sites labeled with R1. (C) Room temperature spectra of sites labeled with V1; the residual free signal has been subtracted. The definition of 2A_zz’ is shown for 134V1 and all values of 2A_zz’ are indicated.

Figure 4

EPR spectrum and simulation of T4L 72V1 attached to CNBr activated Sepharose. The EPR spectrum was recorded at 298K in buffer without sucrose. The best fit (Materials and Methods) was obtained with an isotropic nitroxide diffusion of R =10^8.39 and with coefficients of the ordering potential being C₂₀ = 2.0, C₄₀=2.7; these values give a strong localization of the Z axis of the director along the 2p orbital of the nitroxide.

Figure 5

Influence of nearest neighbor side chains on the motion of 131V1. (A) Model of the H helix of T4L showing the nearest neighbors. (B) EPR spectrum of 131V1 and the corresponding spectra for single alanine substitution mutants at the indicated sites.

Figure 6

Crystal structure of T4L 65V1/76V1 at 100K. (A) Electron density map for V1 and neighboring side chains are shown for the C helix. The electron density (blue mesh) was calculated as an unweighted 2Fo-Fc map contoured at 1.0σ. (B) and (C) Space filling CPK models and electron density maps of 65V1 and 76V1, respectively, showing intra-side chain van der Waals interactions of the ring 3-N atom with the Sγ sulfur. The structure is deposited to the RCSB Protein Data Bank (PDB ID: 3K2R)

Figure 7

Comparison of the interspin distance in 65V1/76V1 determined from the crystal structure and DEER spectroscopy. (A) A model of the C helix containing 65V1 and 76V1, showing the interspin distance determined from the crystal structure. (B) Dipolar evolution functions and (C) derived distance distributions are shown for 65V1/76V in blue. The interspin distance from the crystal structure is shown as a vertical dotted line. For comparison, the data for 65R1/67R1 is shown in red. The best fit dipolar evolution function is overlaid in (B) using the corresponding colors.

Figure 8

Interspin distance distributions determined from DEER for the indicated V1 labeled double mutant and comparison with R1. (A) Dipolar evolution functions and (B) derived distance distributions are shown for 68V1/81V (blue) and 68R1/81R1 (red). The best fit dipolar evolution function is overlaid in (A) using the corresponding colors. (C) T4L with V1 modeled in the {t, p, p, -22°} configuration at sites 68 and 81. The interspin distance from modeling is indicated and agrees closely with the experimental value.