Protein-nucleic acid recognition probed by solid-state Nuclear Magnetic Resonance spectroscopy at fast Magic-Angle Spinning
快速魔角旋转固态核磁共振波谱探测蛋白质-核酸识别
基本信息
- 批准号:455240421
- 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:
- 资助国家:德国
- 起止时间:
- 项目状态:未结题
- 来源:
- 关键词:
项目摘要
Protein-nucleic acid interactions are involved in a variety of biological events ranging from the replication of genomic DNA to the synthesis of proteins. Noncovalent interactions such as hydrogen bonds, dispersion and electrostatic interactions are responsible for the molecular recognition of nucleic acids by proteins. This project focusses on the detection of such weak chemical interactions in non-crystalline protein-nucleic acid complexes. Solid-state Nuclear Magnetic Resonance (NMR) methods (the “NONCOV” approach) will be developed and implemented to probe such effects directly, particularly focussing on protons which are at the centre of protein-nucleic acid contacts. Proton NMR observables serve as sensitive reporters for the engagement of protons in such contacts and their measurement will be achieved by fast Magic-Angle spinning (MAS) experiments (employing MAS frequencies of more than 100 kHz) allowing for a sufficient reduction of the proton NMR linewidths. In combination with the experimental data, quantum-chemical calculations of NMR observables carried out on small protein-nucleic acid fragments will lead to an increase of the theoretical understanding of the response of an NMR observable to the strength of a specific noncovalent interaction. The final objective of NONCOV is to model the binding of nucleic acid to large protein assemblies. Therefore, hydrogen bonds between the protein and RNA/DNA phosphate groups will be identified in proton-detected 1H,31P correlation experiments. Besides restraints based on the direct responses of proton NMR observables (e.g. proton chemical-shift values and J-coupling constants) on protein-RNA/DNA binding events, distance restraints from paramagnetic NMR experiments will be extracted by attaching paramagnetic spin labels to nucleic acids and by investigating paramagnetic relaxation enhancements on the protein NMR spectra. This will allow us additionally to determine distance restraints by Electron Paramagnetic Resonance experiments, for example between a spin label bound to the nucleic acid and a tag attached to the protein. As a proof-of-principle, the NONCOV approach will be established for the bacterial DnaB helicase from Helicobacter pylori involved in unwinding double-stranded DNA during DNA replication. This will enable further insights into the conformational and dynamic changes occurring during DNA loading and translocation of such ring-shaped helicases. The NONCOV approach is transferrable to further biological and chemical applications in which noncovalent interactions are involved, e.g. in the context of phase separation phenomena or supramolecular chemistry.
蛋白质-核酸相互作用涉及从基因组DNA复制到蛋白质合成的各种生物学事件。蛋白质对核酸的分子识别是由氢键、弥散和静电相互作用等非共价相互作用引起的。本项目致力于检测非晶态蛋白质-核酸复合体中这种微弱的化学相互作用。将开发和实施固态核磁共振方法(“NONCOV”方法),以直接探测这种影响,特别是关注处于蛋白质-核酸接触中心的质子。质子核磁共振观测数据是质子在这种接触中接触的灵敏报告,它们的测量将通过快速魔角旋转(MAS)实验(使用超过100 kHz的MAS频率)来实现,从而充分减少质子核磁共振线宽。结合实验数据,对蛋白质-核酸小片段进行的核磁共振观测的量子化学计算将增加对可观测到的核磁共振对特定非共价相互作用强度的响应的理论理解。NONCOV的最终目标是模拟核酸与大型蛋白质组合的结合。因此,蛋白质和RNA/DNA磷酸基团之间的氢键将在质子检测的1H,31P关联实验中得到鉴定。除了基于质子核磁共振可观测物对蛋白质-RNA/DNA结合事件的直接响应(例如质子化学移动值和J偶合常数)的约束外,还将通过将顺磁自旋标记附着在核酸上并通过研究顺磁驰豫对蛋白质核磁共振谱的增强来提取来自顺磁核磁共振实验的距离约束。这将使我们能够通过电子顺磁共振实验来确定距离限制,例如,结合到核酸上的自旋标记和附着在蛋白质上的标记之间的距离限制。作为一项原则证明,将为幽门螺杆菌的细菌DNAB解旋酶建立NONCOV方法,该酶在DNA复制过程中参与解开双链DNA。这将使人们能够进一步深入了解在DNA装载和转位过程中发生的构象和动态变化。NONCOV方法可转移到涉及非共价相互作用的进一步生物和化学应用中,例如在相分离现象或超分子化学的背景下。
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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Professor Dr. Thomas Wiegand其他文献
Professor Dr. Thomas Wiegand的其他文献
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{{ truncateString('Professor Dr. Thomas Wiegand', 18)}}的其他基金
Cellular organization by phase separation processes investigated by solid-state NMR: Insights from RNA-binding proteins and engineered spider silk proteins
通过固态 NMR 研究相分离过程的细胞组织:RNA 结合蛋白和工程蜘蛛丝蛋白的见解
- 批准号:
455238107 - 财政年份:
- 资助金额:
-- - 项目类别:
Heisenberg Grants
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