Phytochromes: Structural Perspectives on Photoactivation and Signaling

项目摘要 大多数生物体采用一系列光感受器来检测它们的光环境。可以说，最有影响力的是 光敏色素（Phys）是植物生长发育所必需的一个多样性类群，广泛分布于许多植物中， 细菌、真菌和藻类属。通过它们的胆色素发色团在红光- 吸收Pr态和吸收远红光的Pfr态，Phys在各种信号级联中充当光开关 对光的强度、方向、持续时间和光谱质量敏感。此外，通过Pfr的热回复， 对于Pr，一些Phys通过对该反应速率的双折射效应来感知温度，并且可能通过 Pfr的夜间消耗。这种Pr/Pfr相互转换的累积效应影响了许多生理学上的变化。 对农业以及有害植物和人类病原体的生物学具有重要意义的过程。此外，其独特的 光化学最近提供了宝贵的光遗传学工具，包括用于组织成像的新荧光团， 以及可以在时间和空间上精确调控细胞活动的工程光开关。 最近，我们和其他人通过对光敏的研究，在理解物理信号方面取得了很大进展。 地区一个新兴的toggle模型假定，光引发的胆色素异构化产生了埃级 胆色素结合口袋内的重排，最终转化为大规模的构象变化， 二聚体光感受器。虽然该模型有助于澄清最终状态转换所需的总体更改， 中间体的光激发和随之而来的结构变化所必需的一个信号主管Pfr状态是 不确定鉴于其独特的模块化架构，目前还不清楚该模型适用于植物Phys的效果如何。 本提案的目的是通过对以下物质的持续结构和生物化学分析， 在一些实施方案中，将代表性Phys与它们的Pr和Pfr状态组合，并与它们的下游效应物组合。具体目标是： (1)利用X射线晶体学和低温电子显微镜研究更全面的植物结构， 细菌Phys，包括全长二聚体光感受器及其信号输出模块的模型;（2）定义如何 物理学通过与其下游伙伴的关联对光信号进行处理;（3）利用串行飞秒x射线 晶体学在结构上定义光子吸收后产生的中间体;（4）使用稳态和表面 映射方法，以更好地了解蛋白质表面动力学在光转换;和（5）欣赏如何 植物Phy家族内的多样性用于通过生物化学和结构增强热感知。 PhyB同种型的分析，该同种型在其N-末端采用预测的内在无序区域来感测温度。 总之，这个项目将提供一个必要的框架，以更好地了解结构，变构机制， 和Phy超家族的进化。其预期的结果应该有助于阐明微生物和植物如何感知 光，温度，可能还有时间，这可能对改善农业生产有重要影响。 作物的性能，了解微生物生态系统，控制医学相关的生命周期， 病原体，并加强Phys作为光遗传学试剂的应用。

PROJECT SUMMARY Most organisms employ an array of photoreceptors to detect their light environment. Arguably the most influential are the phytochromes (Phys), a diverse group essential for plant growth and development, and widely distributed in many bacterial, fungal, and algal genera. By reversible photointerconversion of their bilin chromophores between a red light- absorbing Pr state and a far-red light-absorbing Pfr state, Phys act as photoswitches in various signaling cascades responsive to light intensity, direction, duration, and spectral quality. Moreover, through the thermal reversion of Pfr back to Pr, some Phys sense temperature through enthalpic effects on the rate of this reaction, and possibly perceive time via the nighttime depletion of Pfr. The cumulative effects of this Pr/Pfr interconversion impact numerous physiological processes important to agriculture and the biology of harmful plant and human pathogens. In addition, their unique photochemistries have recently provided invaluable optogenetic tools, including novel fluorophores for tissue imaging, and engineered photoswitches that can regulate cellular events with remarkable temporal and spatial precision. Recently, we and others have made great strides in understanding how Phys signal through studies on the photosensing region. An emerging toggle model posits that a light-triggered isomerization of the bilin yields angstrom-scale rearrangements within the bilin-binding pocket that is ultimately transduced into large-scale conformational changes in the dimeric photoreceptor. While the model helps clarify gross changes required for endstate conversion, the intermediates of photoexcitation and ensuing structural changes necessary for a signaling-competent Pfr state are uncertain. It is also unclear how well the model applies to plant Phys given their distinctive modular architectures. The objective of this proposal is to complete this picture through continued structural and biochemical analyses of representative Phys in their Pr and Pfr states, and in combination with their downstream effectors. Specific aims are to: (1) use x-ray crystallography and cryo-electron microscopy to develop more comprehensive structures of plant and bacterial Phys, including models of full-length dimeric photoreceptors with theirs signal output modules; (2) define how Phys transduce the light signal through association with their downstream partners; (3) exploit serial femtosecond x-ray crystallography to structurally define the intermediates generated after photon absorption; (4) use steady-state and surface mapping methods to better understand the protein surface dynamics during photoconversion; and (5) appreciate how diversity within the plant Phy family is used to enhance thermal perception through the biochemical and structural analyses of the PhyB isoform that employs a predicted intrinsically disorder region at its N-terminus to sense temperature. Taken together, this project will provide an essential framework to better appreciate the structure, allosteric mechanism, and evolution of the Phy superfamily. Its anticipated results should help elucidate how microorganisms and plants sense light, temperature, and possibly time, which could have important ramifications for improving the agricultural performance of crop plants, understanding microbial ecosystems, controlling the life cycle of medically-relevant pathogens, and enhancing the application of Phys as optogenetic reagents.