Protein-nucleic acid recognition probed by solid-state Nuclear Magnetic Resonance spectroscopy at fast Magic-Angle Spinning

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事件。非共价相互作用，如氢键、分散和静电相互作用是蛋白质对核酸分子识别的原因。本项目的重点是检测非晶体蛋白-核酸复合物中这种弱化学相互作用。固态核磁共振（NMR）方法（“NONCOV”方法）将被开发和实施，以直接探测这种影响，特别是关注位于蛋白质-核酸接触中心的质子。质子核磁共振可观测值作为质子在这种接触中的参与的敏感报告，它们的测量将通过快速的Magic-Angle spinning （MAS）实验（使用超过100 kHz的MAS频率）来实现，允许质子核磁共振线宽的充分减少。结合实验数据，对小蛋白质-核酸片段进行核磁共振可观测物的量子化学计算将增加对核磁共振可观测物对特定非共价相互作用强度响应的理论理解。NONCOV的最终目标是模拟核酸与大蛋白组装体的结合。因此，在质子检测1H，31P相关实验中，蛋白质和RNA/DNA磷酸基团之间的氢键将被识别。除了基于质子核磁共振观测值（如质子化学位移值和j偶联常数）对蛋白质- rna /DNA结合事件的直接响应的约束外，顺磁核磁共振实验的距离约束将通过在核酸上附加顺磁自旋标签和研究顺磁弛豫对蛋白质核磁共振波谱的增强来提取。这将使我们能够通过电子顺磁共振实验来确定距离限制，例如与核酸结合的自旋标签和附着在蛋白质上的标签之间的距离限制。作为原理证明，NONCOV方法将建立在幽门螺杆菌中参与DNA复制过程中双链DNA解绕的细菌DNA解旋酶。这将有助于进一步了解这种环状解旋酶在DNA装载和易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。

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.