Rohmer Thierry, Strauss Holger, Hughes Jon, de Groot Huub, Gärtner Wolfgang, Schmieder Peter, Matysik Jörg
Leiden Institute of Chemistry, Gorlaeus Laboratoria, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands.
J Phys Chem B. 2006 Oct 19;110(41):20580-5. doi: 10.1021/jp062454+.
Solid-state nuclear magnetic resonance (NMR) is applied for the first time to the photoreceptor phytochrome. The two stable states, Pr and Pfr, of the 59-kDa N-terminal module of the cyanobacterial phytochrome Cph1 from Synechocystis sp. PCC 6803 containing a uniformly 15N-labeled phycocyanobilin cofactor are explored by 15N cross-polarization (CP) magic-angle spinning (MAS) NMR. As recently shown by 15N solution-state NMR using chemical shifts [Strauss, H. M.; Hughes, J.; Schmieder, P. Biochemistry 2005, 44, 8244], all four nitrogens are protonated in both states. CP/MAS NMR provides two additional independent lines of evidence for the protonation of the nitrogens. Apparent loss of mobility during photoactivation, indicated by the decrease of line width, demonstrates strong tension of the entire chromophore in the Pfr state, which is in clear contrast to a more relaxed Pr state. The outer rings (A and D) of the chromophore are significantly affected by the phototransformation, as indicated by both change of chemical shift and line width. On the other hand, on the inner rings (B and C) only minor changes of chemical shifts are detected, providing evidence for a conserved environment during phototransformation. In a mechanical model, the phototransformation is understood in terms of rotations between the A-B and C-D methine bridges, allowing for intramolecular signal transduction to the protein surface by a unit composed of the central rings B and C and its tightly linked protein surroundings during the highly energetic Pfr state.
固态核磁共振（NMR）首次应用于光感受器光敏色素。通过15N交叉极化（CP）魔角旋转（MAS）NMR研究了来自集胞藻属PCC 6803的蓝藻光敏色素Cph1的59 kDa N端模块的两种稳定状态Pr和Pfr，该模块含有均匀15N标记的藻胆青素辅因子。正如最近通过使用化学位移的15N溶液态NMR所表明的[施特劳斯，H. M.；休斯，J.；施密德，P.《生物化学》2005年，44卷，8244页]，在两种状态下所有四个氮原子都被质子化。CP/MAS NMR为氮原子的质子化提供了另外两条独立的证据。光激活过程中明显的迁移率损失，由线宽的减小表明，证明了Pfr状态下整个生色团的强烈张力，这与更松弛的Pr状态形成鲜明对比。生色团的外环（A和D）受到光转化的显著影响，化学位移和线宽的变化都表明了这一点。另一方面，在内环（B和C）上仅检测到化学位移的微小变化，这为光转化过程中保守的环境提供了证据。在一个力学模型中，光转化被理解为A - B和C - D次甲基桥之间的旋转，在高能的Pfr状态下，由中央环B和C及其紧密相连的蛋白质环境组成的单元允许分子内信号传导至蛋白质表面。
