Schmidt Marius, Pahl Reinhard, Srajer Vukica, Anderson Spencer, Ren Zhong, Ihee Hyotcherl, Rajagopal Sudarshan, Moffat Keith
Physikdepartment E17, Technische Universität München, 85747 Garching, Germany.
Proc Natl Acad Sci U S A. 2004 Apr 6;101(14):4799-804. doi: 10.1073/pnas.0305983101. Epub 2004 Mar 23.
We determine the number of authentic reaction intermediates in the later stages of the photocycle of photoactive yellow protein at room temperature, their atomic structures, and a consistent set of chemical kinetic mechanisms, by analysis of a set of time-dependent difference electron density maps spanning the time range from 5 micros to 100 ms. The successful fit of exponentials to right singular vectors derived from a singular value decomposition of the difference maps demonstrates that a chemical kinetic mechanism holds and that structurally distinct intermediates exist. We identify two time-independent difference maps, from which we refine the structures of the corresponding intermediates. We thus demonstrate how structures associated with intermediate states can be extracted from the experimental, time-dependent crystallographic data. Stoichiometric and structural constraints allow the exclusion of one kinetic mechanism proposed for the photocycle but retain other plausible candidate kinetic mechanisms.
通过分析一组时间范围从5微秒到100毫秒的时间相关差分电子密度图，我们确定了室温下光活性黄色蛋白光循环后期真实反应中间体的数量、它们的原子结构以及一组一致的化学动力学机制。将指数成功拟合到从差分图的奇异值分解得到的右奇异向量上，表明化学动力学机制成立且存在结构不同的中间体。我们识别出两个与时间无关的差分图，从中细化了相应中间体的结构。因此，我们展示了如何从实验性的、与时间相关的晶体学数据中提取与中间态相关的结构。化学计量和结构约束允许排除一种为光循环提出的动力学机制，但保留其他合理的候选动力学机制。
