Heme-based sensor kinases in Methanosarcina acetivorans as a paradigm for archaeal signal transduction
乙酰甲烷八叠球菌中基于血红素的传感器激酶作为古菌信号转导的范例
基本信息
- 批准号:259019293
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:德国
- 项目类别:Research Grants
- 财政年份:2014
- 资助国家:德国
- 起止时间:2013-12-31 至 2018-12-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
The methyl sulfide methyltransferase associated Sensor MsmS (MA4561) from the methanogenic archaeon Methanosarcina acetivorans is a multidomain sensor protein consisting of alternating PAS and GAF domains joint to a C-terminal histidine kinase-like H_ATPase output domain. MsmS contains a covalently bound heme-cofactor in its second GAF domain. This heme is redox active and has the ability to ligate several external ligands including carbon monoxide and dimethyl sulfide. Upon oxidation of the heme cofactor autophosphorylation activity of the H_ATPase domain is induced suggesting that MsmS is a sensor kinase. With the help of a chromosomal knock out mutant of msmS we could further show that MsmS is involved in the cross-regulation of one of three gene encoding corrinoid/methyltransferase isozymes. The transcription of these genes was shown to be activated by upstream encoded regulators of the Msr family. One of these regulators is encoded downstream of msmS with which it forms a bicistronic operon suggesting that they form a two component system.Within this grant proposal we wish to investigate MsmS and the homologous kinase MA0863 and their role in regulating the corrinoid/methyltransferase isozymes. One line of investigation will characterize the coordination structure of the heme within this prototypical heme sensor kinase from Archaea and specifically the role of the covalent bond will be examined. Preliminary results indicated that MA0863 has also a heme bound in its second GAF domain. It will be investigated whether the redox properties and the axial coordination is different from the heme found in Msms. Therefore, a combination of UV-vis, resonance Raman und magnetic circular dichroism spectroscopy will be employed. Redox potentials will be determined using redox titration experiments. Although both sensors share high amino acid sequence homology, MA0863 is special in that it encodes an amber codon in its second PAS domain which is translated to the amino acid pyrrolysine in M. acetivorans. Whether pyrrolysine serves as a regular amino acid residue or holds a special function is another objective of this proposal. The signal transduction pathways from the sensor kinases and possible cross regulation of the corrinoid/methyltransferase isozymes will be investigated. Besides the transcriptional regulation of the target genes encoding the corrinoid/methyltransferase isozymes, we also wish to explore the function of the Msr regulators in the signal transduction process. This will be done with the help of protein-protein interaction studies between the sensor kinases and the regulator proteins and DNA-regulator interaction studies using gel mobility shift assays. Overall, we wish to contribute to the understanding of archaeal signal transduction in general but on methanogen physiology in particular.
来自产甲烷古菌Methanosarcina acetivorans的甲基硫化物甲基转移酶相关传感器mms (MA4561)是一个多结构域传感器蛋白,由交替的PAS和GAF结构域连接到c端组氨酸激酶样h_atp酶输出结构域。mms在其第二个GAF结构域含有一个共价结合的血红素辅因子。这种血红素具有氧化还原活性,能够连接几种外部配体,包括一氧化碳和二甲基硫化物。在血红素辅助因子氧化后,h_atp酶结构域的自磷酸化活性被诱导,这表明MsmS是一种传感器激酶。在染色体敲除突变体的帮助下,我们可以进一步证明msmS参与编码corcoroid /甲基转移酶同工酶的三个基因之一的交叉调控。这些基因的转录被上游编码的Msr家族调控因子激活。其中一个调节因子编码在msmS的下游,它与msmS形成双电子操纵子,这表明它们形成了一个双组分系统。在这项拨款申请中,我们希望研究MsmS和同源激酶MA0863及其在调节类玉米粉/甲基转移酶同工酶中的作用。一条研究线路将表征血红素的配位结构,在这个原型血红素传感器激酶来自古菌,特别是共价键的作用将被检查。初步结果表明,MA0863在其第二个GAF结构域也有一个血红素结合。将研究其氧化还原性质和轴向配位是否与mms中的血红素不同。因此,将采用紫外-可见、共振拉曼和磁圆二色光谱相结合的方法。氧化还原电位将通过氧化还原滴定实验来确定。虽然这两个传感器都具有很高的氨基酸序列同源性,但MA0863的特殊之处在于,它在其第二PAS结构域编码一个琥珀色密码子,该密码子可翻译为m.a actitivorans的氨基赖氨酸。pyrlysine是作为一种常规的氨基酸残基还是具有特殊的功能是本提案的另一个目标。传感器激酶的信号转导途径和可能的corcoroid /甲基转移酶同工酶的交叉调节将被研究。除了编码椰碱/甲基转移酶同工酶的靶基因的转录调控外,我们还希望探索Msr调控因子在信号转导过程中的功能。这将在传感器激酶和调节蛋白之间的蛋白质-蛋白质相互作用研究和dna -调节相互作用研究的帮助下完成,使用凝胶迁移位移测定。总的来说,我们希望对古细菌信号转导的理解有所贡献,尤其是对产甲烷菌生理学的理解。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Professorin Dr. Nicole Frankenberg-Dinkel其他文献
Professorin Dr. Nicole Frankenberg-Dinkel的其他文献
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{{ truncateString('Professorin Dr. Nicole Frankenberg-Dinkel', 18)}}的其他基金
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