3V3X

Nitroxide Spin Labels in Protein GB1: N8/K28 Double Mutant

Summary for 3V3X

• Entry DOI: 10.2210/pdb3v3x/pdb
• Descriptor: Immunoglobulin G-binding protein G, S-[(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl] methanesulfonothioate, NONAETHYLENE GLYCOL, ... (6 entities in total)
• Functional Keywords: domain-swapped dimer, nitroxide spin label, immune system
• Biological source: Streptococcus sp. 'group G'
• Cellular location: Secreted, cell wall; Peptidoglycan-anchor (Potential): P19909
• Total number of polymer chains: 4
• Total formula weight: 27242.61

Authors

Cunningham, T.F.,McGoff, M.S.,Sengupta, I.,Jaroniec, C.P.,Horne, W.S.,Saxena, S.K. (deposition date: 2011-12-14, release date: 2012-08-29, Last modification date: 2024-10-30)

Primary citation

Cunningham, T.F.,McGoff, M.S.,Sengupta, I.,Jaroniec, C.P.,Horne, W.S.,Saxena, S.
High-resolution structure of a protein spin-label in a solvent-exposed beta-sheet and comparison with DEER spectroscopy.
Biochemistry, 51:6350-6359, 2012

PubMed 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.

PubMed: 22809334
DOI: 10.1021/bi300328w

Experimental method

X-RAY DIFFRACTION (2 Å)