loop区域序列修饰的多肽寡聚体结构和聚集机理的光谱学研究

Proteins can misfold to form highly ordered structure rich in cross-β-sheet, termed as amyloid fibrils. During the early stages of the self-assembly, oligomeric aggregates are observed with highly diverse morphology and evidence has been developed to support their being the pathogenic species underlining amyloid related diseases. Due to the dynamic and transient natures of the oligomers, structural studies require stabilization of the oligomers in prior to analysis. Here we design a series of β-sheet containing peptides with sequence mutations in the loop region where the β-strands are connected. This strategy targets at the reducing the horizontal thickness of the peptide assembly instead of blocking the elongation as reported by literature. Unnatural amino acids such as D-proline and N-substituted Glycines are implanted in the loop region to alter the flexibility and structural zipping ability and thus isolate the oligomers. Isotope edited IR and Vibrational Circular Dichroism techniques combining with theoretical simulations are used to depict the structures of the oligomers at single amino acid resolution and to follow the kinetics of the aggregation with extremely high sensitivity. Early investigation shows that a β-stranded sequence with D-Pro-Gly as loop connection could form an off-pathway oligomer, which was able to convert to fibril like aggregate by thermal perturbation. The method proposed here for obtaining stable oligomer is promising, further investigation towards the aggregation mechanism and toxicity of the peptide oligomers would shine lights on the pathology and drug design for related neurodegenerative diseases.

多肽错误折叠并聚集成以β-交联结构为主的淀粉样纤维的过程与多种神经退行性疾病密切相关；聚集前期生成的寡聚体毒性较大，形貌多样且稳定性低，给结构表征和病理研究增加困难。与文献报道的破坏多肽纤维纵向延伸而稳定寡聚体的策略不同，本申请针对连接β-条带的loop区（转折区）结构柔性大且参与横向聚集的性质，通过改变该区氨基酸的手性、主链骨架或侧链基团来调节其刚性、几何构象或氢键性质以减弱β-折叠片间作用力，破坏横向聚集而获取稳定寡聚体。以同位素标记增强的振动光谱为主进行结构表征，以其对寡聚体结构灵敏度高，可全程跟踪聚集行为。根据本申请而进行的前期研究设计了pG（D-脯氨酸-甘氨酸）修饰loop区的模型多肽并解析了其寡聚体结构，推测为反平行排列双β发卡构象的歧路寡聚体。进一步总结寡聚体“序列-稳定性”规律并推广到疾病相关多肽的结构研究上，将为抗聚集类药物设计提供理论依据。

本项目（21503087）以设计loop区“转折点”为调控策略，研究β-折叠构象模型多肽聚集机理及寡聚体稳定条件，应用于诱导神经退行性疾病相关序列稳定寡聚体的制备及表征，取得了一系列研究进展：.（1）设计合成了D-Pro-Gly为转折的双发卡β-折叠模型多肽，通过荧光内外标手段跟踪其自组装动力学，推断出其聚集过程机理，并以振动圆二色谱分辨其寡聚体及高聚纤维的二级结构区别；.（2）设计合成了两个系列具有同位素13C标记的D-Pro-Gly及Aib-Gly为转折的模型多肽，通过激光温跃红外光谱跟踪了β-折叠构象形成的动力学，得到具有双D-Pro-Gly转折的多肽序列在β-折叠形成速率上快于单D-Pro-Gly转折和侧链疏水协同驱动序列的结论，分析得出D-Pro-Gly作为强力β-转折模块，具有锁定β-折叠结构的功能；研究进一步得出Aib-Gly转折对β-折叠构象稳定作用弱于D-Pro-Gly转折，解析了多条带β-折叠结构的分步动力学常数，为设计神经退行性疾病相关多肽的稳定寡聚体提供了定量参数；.（3）设计合成了不同β-转折修饰的阿兹海默症病理相关多肽片段，通过振动圆二色谱及荧光外标跟踪聚集过程，对比透射电镜形貌表征，得出修饰序列采取和原序列不同聚集路径的结论，进一步设计两亲多肽的凝胶制剂载体，通过pH调控寡聚体和高聚纤维的转化，开发凝胶载药系统。