SEARCHING FOR UNIDENTIFIED EARLY INTERMEDIATES IN THE E46Q PYP PHOTOCYCLE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 盐生盐藻的光活性黄蛋白(PYP)是一种蓝光感受器，其发色团为4-羟基肉桂酸，通过硫代酯键共价连接到Cys69的β-硫上。由于其尺寸小(14 KDa)，且易于产生衍射良好的晶体，PYP已成为研究分子细节信号转导的极佳模型系统。以前对纳秒和较长时间分辨率的PYP单晶的Laue衍射研究(1-4)已经确定了结构上不同的中间物种，这些中间物种可以与观察到的光谱相关联。 在此，我们旨在研究突变体E46Q PYP光循环早期中间产物的结构变化。该突变体是PYP最有趣的突变体之一，因为它在发色团和附近的Glu46残基之间没有氢键，因此它比野生型PYP具有更快的光循环(5-7)。潜在的低温陷阱中间体(8)和E46Q从10 ns到100ms(9，10)的Laue晶体结构动力学表明，由于氢键较弱，低于10 ns的中间体比wt PYP中发现的早期PS中间体更不稳定。这些E46Q的早期中间体也可能比wt-PYP的寿命短，可能在数百皮秒(PS)的范围内。这一假说得到了低温捕获中间体的改进的支持，这表明野生型和E46Q PYP(3，8)中都存在早期中间体和pRCW样物种。 因此，我们建议在过去成功的基础上，提高实验结果的质量，并巩固我们的成果。来自更有效的激光激发和展宽激光脉冲的信噪比提高的新数据应该使我们能够第一次表征初级中间体的结构及其在E46Q PYP光循环期间产生的结构动力学。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Photoactive yellow protein (PYP) from Halorhodospira halophila is a blue-light photoreceptor whose chromophore is a 4-hydroxycinnamic acid which is covalently linked by a thioester bond to the ¿-sulfur of Cys69. Due to its small size (14kDa) and the ease of producing well-diffracting crystals, PYP has served as an excellent model system for studying signal transduction in molecular detail. Previous Laue diffraction studies (1-4) on single PYP crystals with nanosecond (ns) and longer time resolution have identified structurally distinct intermediate species which can be correlated with those observed spectroscopically. Here we aim to study the structural changes of early intermediates in the photocycle of the mutant E46Q PYP. This mutant is one of most interesting mutants of PYP, because it has no hydrogen bonds between the chromophore and the nearby Glu46 residue, thus it is known to have a faster photocycle than the wild type PYP (5-7). The potential cryo-trapped intermediates (8) and the Laue crystallographic structural dynamics from 10 ns to 100 ms (9, 10) of E46Q provides the insight of the sub-10 ns intermediates to be less stable than the early ps intermediates found in wt PYP because of the weaker hydrogen bond. These early intermediates of E46Q might be also expected to have shorter lifetimes than that of wt-PYP, perhaps in the hundreds of picosecond (ps) range. This hypothesis is supported by the refinement of cryotrapped intermediates, which show the early intermediate and pRCW-like species in both wild-type and E46Q PYP (3, 8). Thus we propose to build on our successes in the past, improve the quality of the experimental results, and consolidate our results. New data with improved signal to noise ratio from more efficient laser excitation with stretched laser pulses should allow us to characterize, for the first time, the structure of the primary intermediate and it structural dynamics generated during the E46Q PYP photocycle.