SEARCHING FOR UNIDENTIFIED EARLY INTERMEDIATES IN THE E46Q PYP PHOTOCYCLE

在 E46Q PYP 光循环中寻找未识别的早期中间体

• 批准号: 8171979
• 负责人: HYOTCHERL IHEE
• 金额: $ 2.56万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171979
• 关键词: 4-coumaric acid; Biological Models; Computer Retrieval of Information on Scientific Projects Database; Data; Ectothiorhodospira halophila photoactive yellow protein; Funding; Grant; Hydrogen Bonding; Institution; Lasers; Link; Molecular; Noise; Photoreceptors; Physiologic pulse; Proteins; Research; Research Personnel; Resolution; Resources; Signal Transduction; Source; Stretching; Structure; Sulfur; Time; United States National Institutes of Health; chromophore; improved; insight; interest; mutant; nanosecond; success; thioester

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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 来自Halorhodospira halololila的光活性黄色蛋白（PYP）是一种蓝光光感受器，其发色团是一种4-羟基霉素酸，通过硫酯键将其与Cys69的硫酸盐键共价连接。由于其尺寸很小（14KDA）以及易于产生良好的裂纹晶体的易用性，PYP已成为一种出色的模型系统，用于以分子细节研究信号转导。先前对具有纳米秒（NS）和较长时间分辨率的单个PYP晶体的LAUE衍射研究（1-4）已经鉴定出结构上不同的中间物种，可以与观察到的光谱上观察到的中间物种相关。 在这里，我们旨在研究突变E46Q PYP的光圈早期中间体的结构变化。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居住区之间没有氢键，因此已知它具有比野生型PYP更快的光循环（5-7）。 E46Q的潜在冷冻捕获的中间体（8）和LAUE晶体学结构动力学从10 ns到100 ms（9，10）提供了低于WT PYP中PS早期中间体的稳定性的见解，因为弱氢键较弱。 E46Q的这些早期中间体也可能比WT-PYP的寿命要短，可能在数百个Picsecond（PS）范围内。该假设得到了冷冻中间体的改进，该假设表明了野生型和E46Q PYP中的早期中间和PRCW样物种（3，8）。 我们建议以过去的成功为基础，提高实验结果的质量并巩固我们的结果。具有更有效的激光兴奋的带有拉伸激光脉冲的噪声比的新数据应使我们能够首次表征E46Q PYP光循环期间产生的主要中间体和IT结构动力学的结构。