Kneip C, Schlamann W, Braslavsky S E, Hildebrandt P, Schaffner K
Max-Planck-Institut für Strahlenchemie, Postfach 101365, D-45413, Mülheim, Germany.
FEBS Lett. 2000 Oct 6;482(3):252-6. doi: 10.1016/s0014-5793(00)02069-x.
The 39-kDa fragment of oat phytochrome phyA, obtained by tryptic digestion at the amino acids 65 and 425, was studied by resonance Raman spectroscopy. The parent state P(r) reveals far-reaching similarities with that of the native phytochrome implying that the structures of the tetrapyrrole chromophore and its immediate protein environment are not affected by the proteolysis. However, the resonance Raman spectrum of the final product of the P(r) phototransformation, denoted as P(bl), is more closely related to that of the P(fr) precursor of the native phytochrome, i.e. meta-R(C), rather than to that of P(fr) itself. The resonance Raman spectra indicate a high conformational flexibility of the chromophore in P(bl) so that, unlike in P(fr), the tetrapyrrole rings C and D adopt a largely coplanar conformation. The protein interactions with ring D of the chromophore, which in the native phytochrome stabilize the specific chromophore structure of P(fr), cannot be established in the 39-kDa fragment due to the lack of the major C-terminal part of the protein. These findings, furthermore, support the view that the meta-R(C)-->P(fr) transition is associated with a coupling of chromophore and protein structural changes that represent crucial events for the photoactivation of phytochrome.
通过胰蛋白酶在氨基酸65和425处消化获得的燕麦光敏色素phyA的39 kDa片段，用共振拉曼光谱进行了研究。亲本状态P(r)与天然光敏色素的亲本状态有深远的相似性，这意味着四吡咯发色团及其直接的蛋白质环境的结构不受蛋白水解的影响。然而，P(r)光转化最终产物的共振拉曼光谱，记为P(bl)，与天然光敏色素的P(fr)前体的共振拉曼光谱更密切相关，即间位-R(C)，而不是与P(fr)本身的共振拉曼光谱相关。共振拉曼光谱表明P(bl)中发色团具有高度的构象灵活性，因此，与P(fr)不同，四吡咯环C和D在很大程度上采取共平面构象。由于缺乏蛋白质的主要C末端部分，在39 kDa片段中无法建立与天然光敏色素中稳定P(fr)特定发色团结构的发色团环D的蛋白质相互作用。此外，这些发现支持了这样一种观点，即间位-R(C)→P(fr)转变与发色团和蛋白质结构变化的耦合有关，这些变化是光敏色素光活化的关键事件。
