基于质子检测适用于中高分子量膜蛋白结构测定的MAS NMR新方法研究

Three-dimensional struture determination of membrane proteins is still a challenge for protein structure research. Solid-state NMR can be used to study the structures of membrane proteins in their native phospholipid bilayer environment under physiological conditions directly, and its spectral resolution is independent on the protein molecule weight or multimeric state. However, most solid-state NMR methods to facilitate residue-specific resonance assignment rely on low-sensitivity carbon-13 detection, as a result it is very challenging to apply these experiments to large membrane proteins with very limited number of protein molecules in the lipid containing samples.To expand the applications to larger and more biologically complex systems demands improvement in both experimental sensitivity and resolution.Similar to solution NMR, one critical step in the path toward the final structure determination is to assign resonance signals to specific sites in the amino acid sequence.But so far, no mature and effective high-sensitivity solid-state NMR experiments, like solution NMR, are routinely used to assign sequential backbone of membrane proteins yet.In this project, we will develop a suit of high-sensitivity proton-detection based multi-dimensional soild-state NMR experiments, combining with proton spin system dilution and paramagnetic ion doping methods,to facilitate and accelerate residue-specific resonance assignment. These experiments establish exclusively correlation between amide proton and nitrogen with carbon resonance from either the previous or the present residues, but not both, thus effectively double the spectral resolution.With slight modification, these experiments can be used to detect proton-proton distances as long as 10 ?, which are important to refine the tertiarty protein structure.We expect these new proton-detection based triple resonance to accelerate the chemical shift assignment and distance constraints acquistion for large membrane protein in the solid state effectively and expand the practical molecular-weight limit to 35 kDa with the improved sensitivity and resolution.

中高分子量膜蛋白的三维结构测定是蛋白质结构研究的热点和难题。理论上固体NMR谱的分辨率不受膜蛋白分子量大小的限制,可以研究较高分子量的膜蛋白,具有独特优势。但目前用于蛋白质研究的固体NMR方法主要基于低灵敏度的碳-13检测技术，对于较高分子量的膜蛋白体系,非常耗时，使得该方法失效，因此提高固体NMR探测灵敏度是解决问题的关键之一。本项目拟以高灵敏度质子检测MAS NMR方法为基础，结合质子自旋稀释和顺磁弛豫增强技术，设计新的三维和四维实验，提高固体NMR方法的灵敏度和分辨率，从而建立一组较系统的，适用于中高分子量膜蛋白主链顺序归属和长程距离测量的高分辨高灵敏度多维MAS NMR方法。将MAS NMR方法所能解析的膜蛋白分子量大小的上限提高到35 kDa，探索适用于更高分子量膜蛋白体系的相关方法可能性。预期本项目的顺利实施将有助于提高MAS NMR研究膜蛋白三维结构的能力。

水和作用对于膜蛋白的结构和功能有着非常重要的影响，我们以DAGK磷脂膜复合蛋白（DAGK蛋白重组到DMPC/DMPG磷脂中得到）作为模型体系，利用多种固体核磁共振技术详细研究了含水量对磷脂膜复合蛋白中不同运动区域的影响。结果表明，高度可动区域的大幅度运动在含水量达到20 wt%时开启，并且随着含水量的增加，运动加强（含水量在20wt%-50wt%之间），当含水量超过50wt%后，该区域的运动性不再随含水量的变化而变化；对于运动性较差的区域，磷脂可以看作水与蛋白相互作用的媒介（当磷脂在凝胶相时，该区域的运动性不随含水量的变化而变化，当温度靠近磷脂的凝胶-液晶相变温度时，该区域的运动性由磷脂的运动决定，强烈依赖与水和作用）。磷脂膜复合蛋白一般在含水量达到30wt%时，获得较好的MAS NMR灵敏度和分辨率。.利用多维MAS NMR方法研究了钙依赖性磷脂结合蛋白Annexin B12的微观结构，建立了一组系统的适用于中高分子量的膜蛋白主链化学位移指认和顺序识别的，选择性地，仅检测残基间或残基内化学位移关联的高分辨高灵敏度的多维MAS NMR 方法，将MAS NMR方法所能解析的膜蛋白分子量大小的上限提高到35 kDa。