Integrated analysis of the conformation of a protein-linked spin label by crystallography, EPR and NMR spectroscopy

Abstract

Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein-protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.

Fig. 1

Crystal structure of CylR2 55R1. a Side view; the 55R1 side chain is shown in stick representation for both monomers. b Top view; the distance between the nitroxide oxygens is indicated by a dashed line. c and d Electron density of the stick models of R1 in monomer a and b, respectively. σ-weighted map rendered at 1.0 σ

Fig. 2

Superposition of the R1 side chains from monomer a and b of CylR2 55R1. The superposition is based on the main chain atoms of residues 54–56

Fig. 3

DEER-EPR data. a Background corrected DEER time-domain (black) and after Thikhonov regularization (red). b Distance distribution obtained after Thikhonov regularization

Fig. 4

Comparison of experimental paramagnetic relaxation enhancement observed in 2D ¹H-¹⁵N HSQC NMR spectra of CylR2 55R1 with values back-calculated from the crystal structure of CylR2 55R1. a Experimental (black) and back-calculated (grey) PRE intensity ratios, I_param/I_dia, as a function of residue number. Error bars were obtained from repeat measurements. Decreases in peak intensity ratios that occur far from the site of spin-labelling (>10 residues) are indicative of long-range contacts (<25 Å) between the spin-label and distant areas of sequence. b Comparison of intramolecular distances in the crystal structure of CylR2 55R1 with values calculated from the experimental PRE ratios. Residues, for which the signal was broadened beyond detection (upper distance value of 14 Å), are marked in grey. Black lines indicate ±5 Å

Fig. 5

Influence of the conformation of the MTSL tag on paramagnetic relaxation enhancement in CylR2 55R1. a Theoretical positions of the paramagnetic oxygen in the 3D structure of CylR2 for conformations of the MTSL tag from the T4L structure. Left hand side: oxygen positions C1 (Magenta), C2 (orange), C3 (yellow), C4 (green) and C5 (blue) for the 72R1 conformations listed in Table 1. Right hand side: positions corresponding to 131R1. The oxygen location observed in the crystal structure of CylR2 55R1 is shown in red. Two views related by a 90° rotation are presented. b Comparison of the experimental PRE intensity profile (black) with the pattern calculated for the 131R1 C2 conformation (grey). Error bars were obtained from repeat measurements. Note the large deviation between the two profiles in comparison to Fig. 4