3V3X

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

3V3X の概要

• エントリーDOI: 10.2210/pdb3v3x/pdb
• 分子名称: 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)
• 機能のキーワード: domain-swapped dimer, nitroxide spin label, immune system
• 由来する生物種: Streptococcus sp. 'group G'
• 細胞内の位置: Secreted, cell wall; Peptidoglycan-anchor (Potential): P19909
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 27242.61

構造登録者

Cunningham, T.F.,McGoff, M.S.,Sengupta, I.,Jaroniec, C.P.,Horne, W.S.,Saxena, S.K. (登録日: 2011-12-14, 公開日: 2012-08-29, 最終更新日: 2024-10-30)

主引用文献

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

実験手法

X-RAY DIFFRACTION (2 Å)