SEARCHING FOR UNIDENTIFIED EARLY INTERMEDIATES IN THE E46Q PYP PHOTOCYCLE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 来自嗜盐盐螺旋菌的光活性黄蛋白（PYP）是一种蓝光感光体，其发色团是4-羟基肉桂酸，其通过硫酯键与Cys 69的<$-硫共价连接。由于PYP的小尺寸（14 kDa）和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优秀模型系统。先前在单个PYP晶体上进行的具有纳秒（ns）和更长时间分辨率的劳厄衍射研究（1-4）已经鉴定了结构上不同的中间物质，其可以与光谱上观察到的那些相关联。 在这里，我们的目的是研究早期中间体的结构变化的突变体E46 Q PYP的光循环。该突变体是PYP最令人感兴趣的突变体之一，因为它在发色团和附近的Glu 46残基之间没有氢键，因此已知它具有比野生型PYP更快的光循环（5-7）。潜在的低温捕获中间体（8）和从10 ns到100 ms的劳厄晶体结构动力学（9，10）的E46 Q提供了亚10 ns中间体的洞察，因为较弱的氢键，比在wt PYP中发现的早期ps中间体更不稳定。E46 Q的这些早期中间体也可能具有比wt-PYP更短的寿命，可能在数百皮秒（ps）范围内。这一假设得到了冷冻捕获的中间体的改进的支持，其显示了野生型和E46 Q PYP中的早期中间体和pRCW样物质（3，8）。 因此，我们建议在过去成功的基础上，提高实验结果的质量，巩固我们的成果。新的数据与改进的信噪比，从更有效的激光激发与拉伸激光脉冲应该让我们来表征，第一次，主要中间体的结构和它的结构动力学过程中产生的E46 Q PYP光循环。

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.