Molecular mechanisms of signaling systems responsive to light, redox and chemical environment

信号系统响应光、氧化还原和化学环境的分子机制

• 批准号: 10626098
• 负责人: BRIAN R CRANE
• 金额: $ 73.37万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626098
• 关键词: Architecture; Behavior; Biochemical; Biological Process; Borrelia; Cells; Cellular Assay; Chemicals; Chemistry; Chemoreceptors; Chemotaxis; Cholera; Circadian Rhythms; Coupling; Cryoelectron Microscopy; Defect; Devices; Diabetes Mellitus; Disease; Drosophila melanogaster; Electron Spin Resonance Spectroscopy; Environment; Exhibits; Feedback; Flagella; Flavins; Functional disorder; Gene Expression; Generations; Genes; Genetic Transcription; Goals; Helicobacter pylori; Human; Immune Evasion; Immune system; In Vitro; Infection; Invaded; Investigation; Ions; Light; Lyme Disease; Macromolecular Complexes; Malignant Neoplasms; Manic; Measures; Membrane; Memory; Mental Depression; Mental disorders; Metabolic; Metabolic Diseases; Metabolism; Metals; Methods; Modeling; Molecular; Molecular Conformation; Motor; Nature; Neurospora crassa; Nucleotides; Obesity; Optics; Order Spirochaetales; Output; Oxidation-Reduction; Pathogenicity; Phase; Phosphotransferases; Photochemistry; Physiologic pulse; Protein Engineering; Proteins; Regulation; Repressor Proteins; Roentgen Rays; Rotation; Schizophrenia; Sensory; Signal Transduction; Sleep Disorders; Spectrum Analysis; Structure; Switching Complex; Syphilis; System; Testing; Tissues; Torque; Transcription Coactivator; Treponema pallidum; Ulcer; Vibrio cholerae; X-Ray Crystallography; biophysical techniques; cell growth; cell motility; circadian pacemaker; design; fly; fungus; human pathogen; in vivo; infectious disease treatment; light entrainment; malignant stomach neoplasm; molecular array; molecular dynamics; nanomachine; novel therapeutic intervention; pathogen; physical property; programs; protein-histidine kinase; receptor; reconstitution; response; sensor

The Crane group studies signal transduction systems that respond to or involve photochemistry and redox chemistry. Our overall goal is to understand the behaviors of bacterial chemotaxis and eukaryotic circadian rhythms at the level of molecular reactivity through the study of macromolecular complexes that underlie transmembrane signaling, motility, and gene expression. Chemotaxis has long served as a key system for studying transmembrane signaling, intracellular information transfer, and cell locomotion. Furthermore, many human pathogens that cause diseases, such as cholera, gastric cancer, and Lyme, rely on chemotaxis to establish and sustain infection. The sensory apparatus of chemotaxis displays remarkable sensitivity, dynamic range, and molecular memory. Chemoreceptors, histidine kinases (CheA), and coupling proteins assemble into large molecular arrays, wherein long-range cooperative interactions among components produce highly specific responses that adapt to changing conditions. This proposal continues efforts to understand receptor:kinase assembly, chemoreceptor conformational signaling, and ultimately, CheA regulation. CheA output modulates Nature's consummate nanomachine – the flagella motor. The architecture of the switch complex within the motor will be refined to better understand torque generation and direction switching. A particular focus will be the pathogenic spirochetes, which exhibit asymmetric flagella rotation at their respective cell ends. The second system, circadian clocks, comprises of cell-autonomous timing devices that pace metabolism to the diurnal cycle. Clocks are composed of transcriptional-translational feedback loops (TTFLs) within which repressor proteins inhibit the transcriptional activators of their own genes. Light entrains the clock phase by stimulating photosensors that impinge directly on the TTFLs. In humans, aberrant clock function causes mental illness (sleep disorders, depression, and mania), cell growth deregulation (cancer), and metabolic defects (diabetes and obesity). This project proposes structural and mechanistic investigations of the key light sensor and repressor activities in representative clocks from fungi (Neurospora crassa) and flies (Drosophila melanogaster). A complimentary set of biophysical techniques, including X-ray crystallography, small-angle X-ray scattering, optical spectroscopy, cryo-electron microscopy, and pulse dipolar ESR spectroscopy (PDS), will be applied to accomplish these goals. Biochemical reconstitution that leverages protein engineering to procure key entities will be combined with cellular assays and organismal studies in order to correlate physical properties with biological function. For PDS, new methods for incorporating spin probes that are based on nitroxides, flavins, nucleotides, and metal ions will be deployed for measuring structure and dynamics both in vitro and in vivo. Computational design and molecular dynamics will be used to test and consolidate models. Overall, this program aims to provide a molecular-level understanding for sensing and response through the synergistic application of chemical and biophysical methods.

起重机小组研究响应或涉及光化学和氧化还原的信号转导系统 化学.我们的总体目标是了解细菌趋化性和真核生物昼夜节律的行为 通过研究基础大分子复合物来研究分子反应水平的节律 跨膜信号传导、运动性和基因表达。趋化性长期以来一直是一个关键系统， 研究跨膜信号传导、细胞内信息传递和细胞运动。而且很多 引起疾病的人类病原体，如霍乱、胃癌和莱姆病，依赖于趋化性， 建立和维持感染。趋化性的感觉器表现出显著的敏感性、动态性、 范围和分子记忆化学受体、组氨酸激酶（CheA）和偶联蛋白组装成 大分子阵列，其中组分之间的长程合作相互作用产生高度特异性的 适应不断变化的条件的反应。这项提案继续努力了解受体：激酶 组装，化学感受器构象信号传导，并最终，CheA调节。CheA输出调制 自然界最完美的纳米机器鞭毛马达。电动机内的开关复合体的结构 将被完善，以更好地了解扭矩产生和方向切换。一个特别的重点将是 致病性螺旋体，其在各自的细胞末端表现出不对称的鞭毛旋转。第二 生物钟系统由细胞自主计时装置组成，将新陈代谢与昼夜周期同步。 生物钟由转录-翻译反馈环（TTFL）组成，其中阻遏蛋白 抑制自身基因的转录激活因子。光通过刺激生物钟的相位 直接撞击TTFL的光电传感器。在人类中，异常的生物钟功能会导致精神疾病（睡眠 疾病、抑郁症和躁狂症）、细胞生长失调（癌症）和代谢缺陷（糖尿病和 肥胖症）。本计画提出关键光感测器与阻遏物之结构与机制研究 在来自真菌（粗糙脉孢菌）和果蝇（黑腹果蝇）的代表性时钟中的活性。一 一套免费的生物物理技术，包括X射线晶体学，小角度X射线散射， 光学光谱，低温电子显微镜，和脉冲偶极ESR光谱（PDS），将应用于 实现这些目标。利用蛋白质工程获得关键实体的生化重建 将与细胞测定和生物体研究相结合，以便将物理特性与 生物功能。对于PDS，新的方法结合自旋探针是基于氮氧化物，黄素， 核苷酸和金属离子将用于测量体外和体内的结构和动力学。 计算设计和分子动力学将用于测试和巩固模型。总的来说，该方案 旨在通过协同应用， 化学和生物物理方法。

项目成果

Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome.

• DOI: 10.1016/j.str.2022.03.010
• 发表时间: 2022-06-02
• 期刊: STRUCTURE
• 影响因子: 5.7
• 作者: Lin, Changfan;Schneps, Connor M.;Chandrasekaran, Siddarth;Ganguly, Abir;Crane, Brian R.
• 通讯作者: Crane, Brian R.

Engineered chemotaxis core signaling units indicate a constrained kinase-off state.

• DOI: 10.1126/scisignal.abc1328
• 发表时间: 2020-11-10
• 期刊: Science signaling
• 影响因子: 7.3
• 作者: Muok AR;Chua TK;Srivastava M;Yang W;Maschmann Z;Borbat PP;Chong J;Zhang S;Freed JH;Briegel A;Crane BR
• 通讯作者: Crane BR

Design, Validation, and Application of an Enzyme-Coupled Hydrogen Sulfide Detection Assay.

• DOI: 10.1021/acs.biochem.8b01083
• 发表时间: 2018-12
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: M. Lynch;B. Crane

Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating One Iron Atom to Transform a [3Fe-4S] to a [4Fe-4S] Cluster in Dph1-Dph2.

• DOI: 10.1021/jacs.1c03956
• 发表时间: 2021-06-30
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Zhang Y;Su D;Dzikovski B;Majer SH;Coleman R;Chandrasekaran S;Fenwick MK;Crane BR;Lancaster KM;Freed JH;Lin H
• 通讯作者: Lin H

Interdomain Linkers Regulate Histidine Kinase Activity by Controlling Subunit Interactions.

域间连接子通过控制亚基相互作用来调节组氨酸激酶活性。

• DOI: 10.1021/acs.biochem.2c00326
• 发表时间: 2022-12-06
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Maschmann, Zachary;Chandrasekaran, Siddarth;Chua, Teck Khiang;Crane, Brian R.
• 通讯作者: Crane, Brian R.