蛋白质骨架中无规则回路(loops)结构的理论研究及在酶分子改造中的应用

The irregular loops, α-helixes, β-strands are the three secondary structural units of protein structures. In proteins aroud 47% residues are located in the loops, and the loops are the most active motifs in proteins, responsible for the stabilty, flexibility, and dynamic activity. The 3D structures of biological macromolecules (proteins, RNA, and DNA) are determined by the molecular interactions forces between the structural units. Subsequently, the biological functions of biological molecules are determined by the molecular structures. In addition to the three known interaction forces (hydrogen-bond interaction, electrostatic interaction, and van de Waals interaction), there are several unconventional molecular interaction forces, such as cation-π interactions, polar hydrogen-π (Hp-π) interactions, π-π stocking interactions, and coordinate interactions, which are less familiar to many researchers. In the 20 natural amino acids 11 of them are involved in the polar hydrogen-π interactions. In DAN and RNA the four nucleic acid components (adenine, guanine, cytosine, and thymine) possess aromatic rings and polar hydrogen atoms, which are the Hp-π interaction acceptors and donors. The peptide bond units of protein backbones are also the Hp-π interaction player. The Hp-π interactions play important role in the structures of DNA and RNA, too. Our previous studies revealed that the interaction energies of Hp-π bonds are close to the common hydrogen bond interactions, and possess many unique properties. In protein structures the interaction orientation of common hydrogen bonds is in the parallel direction of the peptide bond π-planes. In contrast, the Hp-π interactions are in the vertical direction between the peptide bond π-planes. The Hp-π interactions are the main supporting force in the protein irregular loops. Four basic structure types of loops in protein structures are identified according to the Hp-π interaction types. In the proposed research project the roles of Hp-π interactions in protein folding will be studies, the computation methods for calculations of the folding energies in protein loops will be developed, and the relationship between structures and biological functions of loops will be illustrated according to the Hp-π interactions. The Hp-π interaction theory and calculation methods, developed in this study, will be used in the enzyme engineering experiments focusing on the structures of loops to improve the bioactivity and properties of enzyme.

无规则回路(loops)与α-螺旋、β-折叠并列，是蛋白质3D结构的二级结构单元。在蛋白质中47%的氨基酸残基在loop结构中，loops是蛋白质结构中最复杂和最活跃的部分，对蛋白质的稳定性、柔韧性和动态活性起重要作用。许多酶分子的催化活性中心氨基酸和药物靶标蛋白的配体结合位点都在loops上，loops往往是酶分子生物技术改造的主要对象。然而至今仍然不清楚是什么分子间作用力支撑着loop的结构。预研发现，一种研究尚不充分的分子间作用力极性氢-π键在蛋白质折叠中起特殊作用，是loops的主要支撑力。按极性氢-π键的类型二肽折叠分为四种基本构型。计划进一步研究极性氢-π键在蛋白质结构和折叠中的作用，根据极性氢-π键的概念探讨loop的结构类型和蛋白质基本构件（protein blocks），发展loop折叠能的快速计算方法，解释蛋白质中loop的结构与功能的关系，预测和指导酶分子改造实验。

用高档量子化学方法深入研究了蛋白质结构的肽链骨架中的作用力， 20种氨基酸侧链之间的相互作用力，以及氨基酸侧链与肽链骨架间的作用力。特别研究了以前较少研究的阳离子-π键作用、极性氢-π键作用、盐桥作用、酰胺桥作用和疏水作用，用量子化学方法系统计算了这些作用力，建立了数据库，并拟合出简单的势能经验函数。在预研的基础上，集中深入研究了肽链骨架中的极性氢-π键作用，即肽链中的π-平面与肽链中的极性氢在垂直方向形成的极性氢-π键，用高档量子化学方法CCSD(T)计算了肽链骨架中的极性氢-π键的能量与两个二面角（Ψ,Φ）的势能函数，绘制了精确的势能面图。在（Ψ,Φ）势能面上除α-螺旋和β-带外，发现了四个无规则loop结构的基本类型，作为研究肽链折叠的依据。分析了极性氢-π键在蛋白质骨架折叠中的作用，及氨基酸侧链对势能面的影响。深入研究了α-淀粉酶和普鲁兰酶中的无规则loop结构中的极性氢-π键作用，分析了loop结构的热稳定性问题，用于预测蛋白质分子的热稳定性和指导酶分子改造实验。以本研究项目获得的蛋白质内的各种作用力的能量数据为参数，建立了研究蛋白质结构与性质关系的氨基酸序列及理化性质的双层构效关系（2L-QSAR），和蛋白质序列位点及氨基酸理化性质的双层主成分分析法（2L-PCA）。在双层主成分分析法（2L-PCA）中构造了氨基酸理化性质的主成分向量的正交空间和蛋白质残基的序列位点的主成分向量的正交空间.在这两个相互嵌套的正交空间中，蛋白质家族的每一个成员是正交空间里的一个向量，蛋白质在两个正交空间的主成分分量上的投影为分析两类参数对酶的活性的影响提供了数量依据。提出了改进酶分子催化反应的pH值适应性的“活性氢键网络法”和改进酶分子热稳定性的“蛋白质模拟热探测法”，用于α-淀粉酶和普鲁蓝酶的氨基酸定点变异改造，在酶的热稳定性和pH适应性方面得到了正面的改善结果。