High-Resolution Structure of a Protein Spin-Label in a Solvent-Exposed β-Sheet and Comparison with DEER Spectroscopy

Abstract

X-ray crystallography has been a useful tool in the development of site-directed spin labeling by resolving rotamers of the nitroxide spin-label side chain in a variety of α-helical environments. In this work, the crystal structure of a doubly spin-labeled N8C/K28C mutant of the B1 immunoglobulin-binding domain of protein G (GB1) was solved. The double mutant formed a domain-swapped dimer under crystallization conditions. Two rotameric states of the spin-label were resolved at the solvent-exposed α-helical site, at residue 28; these are in good agreement with rotamers previously reported for helical structures. The second site, at residue 8 on an interior β-strand, shows the presence of three distinct solvent-exposed side-chain rotamers. One of these rotamers is rarely observed within crystal structures of R1 sites and suggests that the H(α) and S(δ) hydrogen bond that is common to α-helical sites is absent at this interior β-strand residue. Variable temperature continuous wave (CW) experiments of the β-strand site showed two distinct components that were correlated to the rotameric states observed in crystallography. Interestingly, the CW data at room temperature could be fit without the use of an order parameter, which is consistent with the lack of the H(α) and S(δ) interaction. Additionally, double electron electron resonance (DEER) spectroscopy was performed on the GB1 double mutant in its monomeric form and yielded a most probable interspin distance of 25 ± 1 Å. In order to evaluate the accuracy of the measured DEER distance, the rotamers observed in the crystal structure of the domain-swapped GB1 dimer were modeled into a high-resolution structure of the wild type monomeric GB1. The distances generated in the resulting GB1 structural models match the most probable DEER distance within ~2 Å. The results are interesting as they indicate by direct experimental measurement that the rotameric states of R1 found in this crystal provide a very close match to the most probable distance measured by DEER.

Conformational dynamics and distribution of nitroxide spin labels

“…The strong preference for 1/2 mm and tp rotamers observed for surface-exposed helical and loop sites in T4 lysozyme structures PDB:2IGC, PDB:2OU8, PDB:2NTH [119], PDB:2Q9D, PDB:2Q9E [58], and PDB:1ZYT, PDB:2CUU, PDB:3G3V, PDB:3G3X, and PDB:3G3W [59] is also observed in other soluble proteins (CylR2, PDB:2XIU, [120]; GB1, PDB:3V3X [121]) and, in one instance, at a buried site (Spa15, PDB:2XGA, [122]). For exposed residue Asn-8 in a  strand of GB1 (PDB:3V3X) non-rotameric 2 angles of -120.4° and 162° have been found [121].…”

“…For exposed residue Asn-8 in a  strand of GB1 (PDB:3V3X) non-rotameric 2 angles of -120.4° and 162° have been found [121]. As seen in Non-rotameric angles, defined here as dihedral angles deviating by more than 30° from their canonical value are also observed for 2 in loop site Ser-48 of cytochrome P450cam (PDB:4EK1, [123]), but in no instance for 1 or 3.…”

The Double‐Histidine Cu2+‐Binding Motif: A Highly Rigid, Site‐Specific Spin Probe for Electron Spin Resonance Distance Measurements

“…Significant efforts to investigate R1 conformational preferences through X-ray crystallography [3] and computational techniques [4] have helped to address this limitation in part, though the accurate prediction of interspin distances in proteins remains challenging. Much work has also been devoted to the development of alternate protein spin labels that are more rigid than R1.…”

“…For the nitroxide sample, we modeled R1-modified Cys residues into a published crystal structure of wild-type GB1; rotamers were based on a previous crystal structure (Figure 3c). [3e] The observed Cu 2+ – Cu 2+ distance was interpreted on the basis of the X-ray crystal structure of 6H/8H/28H/32H-GB1 crystallized in the absence of copper (Figure 2a). All efforts to derivatize these crystals with copper were unsuccessful; however, force-field minimization at both the helix and sheet sites provided similar histidine rotamers and coordination geometries to those observed in a previously reported crystal structure of Cu 2+ bound to an IDA derivative and two imidazole ligands.…”

Cysteine-Specific Cu2+ Chelating Tags Used as Paramagnetic Probes in Double Electron Electron Resonance

“…Labeling with MTSSL was performed as previously described. 23 Labeling with MTS-EDTA and TETAC were performed similarly except since both tags are water soluble no DMSO was utilized in the labeling process. Removal of the excess chelating tags was performed similar to the procedure for MTSSL.…”