【作者】 周鹏；

【导师】 商志才；

【作者基本信息】 浙江大学 ， 化学， 2011， 博士

【摘要】 按照经典中心法则的定义,生命体内遗传信息是由DNA经RNA流向多肽序列。近年来随着人们对分子生物世界的深入认识,该信息流经途径得到了进一步扩展,即新生肽链需要折叠成具有精密构造的三维结构,进而与其他生物分子和／或生物基质发生时空特异性的识别和相互作用,最终实现遗传信息从基因型到表型的映射。可以看到,上述扩增部分所涉及到的关键环节即蛋白质折叠和识别是由可逆的弱化学力所支配的,通常称这些化学力为生物非键作用(biological noncovalent interaction)。本文进一步扩展了生物非键作用的概念,用以描述生物环境中一个或数个非键作用按照一定空间排列模式构成的具有一定功能的有机整体,称之为生物非键模体(biological noncovalent motif)。在此基础上,本文对生物分子中几类长期被人们所忽视的非键模体的结构和性质开展了系统的调查；另外还定义了数种新型生物非键模体,并以理论计算和数据库检索相结合的方式论证了它们的存在性和功能性；最后介绍了一种我们开发的用于在二维页面上示意性描绘蛋白质复合物界面非键模体的分子图形学软件包2D-GraLab。下面对这些工作逐一进行概述：(1)蛋白质中的含硫氢键(sulfur-containing hydrogen bond):蛋白质中氮和氧原子参与形成的常规氢键曾引起了相关领域研究者的广泛兴趣并进行了深入研究,但由另一类元素即硫所形成的含硫氢键却长期被人们所忽视了。本文对Top500数据库中所有高质量蛋白质晶体结构进行了统计分析,从中获得了大量关于蛋白质含硫氢键的几何和分布信息。研究发现：①含硫氢键的键长通常大于常规氢键、而键角则普遍小于常规氢键,从而表现出较弱的化学力特征。②Met和Cys的硫原子是不良的氢键受体,但是Cys巯基却是中等强度的氢键供体。③处于复杂蛋白质环境中含硫氢键的几何行为更接近于自由状态下小分子系统氧原子——而非硫原子——所成氢键情况。④在某些情况下含硫氢键对蛋白质结构和功能起着重要的作用,如它们可以促成a螺旋、绑定链间残基、微调局部结构、辅助二硫键生成等。⑤Cys巯基可与芳香性p电子形成较为松散的氢键,其键长大于普通含硫氢键,且其巯基氢原子往往正对于芳香环边缘。(2)蛋白质／配基界面上的氟键(fluorine bond):氟化是当今制药工业常用的一种用于改善先导实体(lead entity)药代动力学性质的手段,然而含氟药物分子亦可通过共价氟原子与靶标蛋白的各类基团发生多样的非键作用以调理二者识别的特异性和亲和力。本文将这些由氟原子直接参与形成的非键作用称为氟键,进而系统调查了大量存在于蛋白质／配基界面上的氟键的几何分布状态以及它们对配基结合的自由能贡献情况。结果表明：①氟键在几何和热力学上表现出类似于上述含硫氢键的表观特征,而在静电机制方面则更接近于卤键而非氢键。②先前他人研究中多次强调的含氟正交多极作用(C-F…C=O)在本文对蛋白质结构数据库(PDB)检索中并不十分突出；与之相对的是,先前多被人们所忽视的非极性氢参与的氟键(C-F…H-C)却被大量发现于蛋白质／配基界面。③MPWLYP泛函和MMFF94力场可以较好地重现高水平氟键键能,故它们可以被考虑用来处理复杂生物氟键系统。④尽管孤立氟键一般较弱,但氟化亦可通过间接的电子效应来影响配基对受体的识别行为。(3)生物分子中的卤-水-氢桥(halogen-water-hydrogen bridge):业已知道,生物环境中水分子可以通过多重氢键网络与周围基质发生相互作用,从而形成水中介的氢键桥系统。我们进而推测与氢键在能量和方向上具有极高相似性的卤键可以功能性地取代水中介氢键桥中的一个或数个氢键,从而形成我们称之为卤-水-氢桥的生物非键模体。在此基础上,本文通过理论分析和晶体结构检索相结合的方式证实了上述猜想,即这种推定的卤-水-氢桥确实存在于生物分子世界,尽管它们并不十分常见。同时本文还借助数个典型实例指出这些观察到的卤-水-氢桥可以显著影响生物分子结构模式,并功能性调理蛋白质／蛋白质、蛋白质／核酸以及蛋白质／药物配基之间的特异性识别和相互作用。(4)生物分子中的卤离子桥(halogen-ionic bridge):生物分子中由电中性的水分子和电正性的金属离子分别介导形成的水中介氢键桥和金属配位桥已经得到了生物化学领域的广泛认同和深入研究,由此我们很自然地联想到普遍存在于生理环境的电负性卤素离子是否亦能参与非键桥联系统,并发挥特异性的生物效应?本文将这类由结构化卤离子所介导的桥联系统命名为卤离子桥以强调其与水分子桥和金属离子桥在结构和功能上的相似性。进而我们通过对小的模型系统、真实的生物分子系统以及PDB数据库的详尽理论分析和统计调查有力确证了该类推测的卤离子桥联模体广泛存在于生物分子世界且在诸多情况中发挥了重要的生物学功能。研究还发现卤素离子可以通过离子氢键、离子键、甚至共价键等方式与生物基团发生有效作用,且这些作用的强度显著大于多数见诸于生物体系的常规非键形式。(5)蛋白质的卤素模体halide motif):卤素模体可视为对卤离子桥的推广它泛指一切迁移到生物分子内部且被固定于亲卤位点的结构化卤离子,以及周围与该离子发生直接或间接非键作用的生物分子基团所构成的一个功能整体。在此基础上本文提出了能量分解分析法energy decomposition analysis)对782个高质量的蛋白质卤素模体所包含的能量成分进行了系统探索,结果发现结构化的卤素模体往往起到稳定蛋白质建筑的作用,且其稳定化自由能贡献与包埋程度成正相关趋势。出乎意料的是,带有一个形式电荷的卤离子主要是通过色散力——而非静电力——来发挥其稳定化效应。这可解释为尽管卤离子可与周围蛋白质基团发生显著的Coulomb吸引作用,但由此获得的有利静电自由能贡献却因形成卤素模体时所伴随的不利静电去溶剂化惩罚而大打折扣了(6)二维描绘蛋白质复合物界面非键模体：有鉴于蛋白质／蛋白质相互作用在诸多生理和生化过程中扮演了中心的角色,本文为此专门开发了一种二维分子图形学软件包2D-GraLab,用于示意性描绘已知结构蛋白质复合物界面的多样非键模体。该程序提供了图形界面、控制对话框、三维显示窗口等交互方式以便用户使用,并借助一系列业已得到相关领域广泛认可的计算技术和独立程序进行数据处理以确保输出结果的可靠性,进而将所得信息以二维示意图方式加以直观呈现以供研究者观察和分析目标蛋白复合物界面的各类非键作用模式。更多还原

【Abstract】 The classic view of the central dogma of biology states that "the coded genetic information hard-wired into DNA is transcribed into individual transportable cassettes, composed of messenger RNA (mRNA); each mRNA cassette contains the program for synthesis of a particular protein sequence." With the progress in the field of biology at subcellular level in recent years, however, our notion about the complete pathway of the genetic information-flow is enlarged to cover biomolecular folding and recognition—that are fundamentally dominated by weak, reversible chemical forces, or called biological noncovalent interactions (BNIs). Here, we further extended the concept of BNI to biological noncovalent motif (BNM) as a description of the functional entity composed of one or several BNIs in biological context. In this dissertation, we define, analyze, and demonstrate a series of specific BNMs that are largely underappreciated in the sophisticated biochemical community. In addition, an in-house program called the 2D-GraLab is also described for automatically generating schematic representation of BNMs across the protein binding interfaces.(ⅰ) Sulfur-containing hydrogen bonds in proteins. Sulfur atoms have been known to participate in hydrogen bonds (H-bonds) and these sulfur-containing H-bonds (SCHBs) are suggested to play important roles in certain biological processes. This study aims to comprehensively characterize all the SCHBs in 500 high-resolution (<1.8 A) protein structures retrieved from the Top500 database. We categorize SCHBs into six types according to donor/acceptor behaviors and then employ explicit hydrogen approach to distinguish SCHBs from those of non-hydrogen bonding interactions. It is revealed that sulfur atom is a very poor H-bond acceptor, but a moderately good H-bond donor. In a-helix, considerable SCHBs are found between the sulphydryl group of cysteine residueⅰand the carbonyl oxygen of residueⅰ-4, and these SCHBs exert appreciable effect in stabilizing helices. Although for most SCHBs, they possess no specific secondary structure preference, their geometrical characteristics in proteins and in free small compounds are significantly distinct, indicating that protein SCHBs are geometrically distorted. Interestingly, sulfur atom in disulfide bond tends to form bifurcated H-bond whereas in cysteine-cysteine pairs prefer to form dual H-bond. These special H-bonds remarkably boost the interaction between H-bond donor and acceptor. By oxidation/reduction manner, the mutual transformation between the dual H-bonds and disulfide bonds for cysteine-cysteine pairs can accurately adjust the structural stability and biological function of proteins in different environments. Furthermore, few loose H-bonds are observed to form between the sulphydryl groups and aromatic rings, and in these cases the donor H is almost over against the rim rather than the center of the aromatic ring.(ⅱ) Fluorine bonds at protein-ligand interfaces. Although fluorination of pharmacologically active compounds has long been a common strategy to increase their metabolic stability and membrane permeation, the functionality of protein-ligand interactions involving fluorine atoms, that we named fluorine bonds, was only recently recognized in chemistry and biology communities. Here, the geometrical characteristics and energetic behaviors of fluorine bonds are systematically investigated by combining two quite disparate but complementary approaches:X-ray structural analysis and theoretical calculations. We find that the short contacts involving fluorine atoms between proteins and fluorinated ligands are very frequent and these contacts, compared to those of routine hydrogen/halogen bonds, are more similar to above-mentioned SCHBs. ONIOM-based QM/MM analysis further reveal that fluorine bonds do play an essential role in protein-ligand binding, albeit the strength of isolated fluorine bonding is quite modest. Furthermore, 14 quantum mechanics (QM) and molecular mechanics (MM) methods are performed to reproduce fluorine bond energies obtained at the rigorous MP2/aug-cc-pVDZ level of theory, and results show that most QM methods and very few MM methods perform well in the reproducibility; the MPWLYP functional and MMFF94 force field are recommended to study moderate and large fluorine bonding systems, respectively.(ⅲ) Halogen-water-hydrogen bridges in biomolecules. The importance of water in biological systems has long been recognized in the field of biology. Here, we describe a new manner by which water affects biomolecular behaviors, called halogen-water-hydrogen bridge (XWH bridge), that is, one H-bond in water-mediated H-bond bridge is replaced functionally by a halogen bond (X-bond). Although behaving similarly to water-mediated H-bond motif, the XWH bridge usually stands in multifurcated forms and possesses stronger directionality. QM analysis on several model and real systems reveals that the XWH bridges are more thermodynamically stable than other water-involved interactions, and this stability is further enhanced by the cooperation between X-bond and H-bond. Crystal structure survey clearly demonstrates the significance of XWH bridges in stabilization of biomolecular conformations and in mediation of protein-protein, protein-nucleic acid, and receptor-ligand recognition and binding.(ⅳ) Halogen-ionic bridges in biomolecules. If considering that the pronouncedly charged halide anions are ubiquitous in the biological world, then it is interesting to ask whether the halogen-ionic bridges—this term is named by us to describe the interaction motif of a nonbonded halogen ion with two or more electrophiles simultaneously—commonly exist in biomolecules and how they contribute to the stability and specificity of biomolecular folding and binding? To address these problems, we herein present a particularly systematic investigation on the geometrical profile and energy landscape of halogen ions interacting with and bridging between polar and charged molecular moieties in small model systems and real crystal structures, by means of ab initio calculation, database survey, continuum electrostatic analysis, and hybrid QM/MM examination. All of these unequivocally demonstrate that this putative halide motif is broadly distributed in biomolecular systems (>6000) and can confer a substantial stabilization for the architecture of proteins and their complexes with nucleic acids and small ligands. This stabilization energy is estimated to be generally more than 100 kcal·mol-1 for gas-phase state or about 20 kcal·mol-1 for solution condition, which is much greater than that found in sophisticated water-mediated bridge (<10 kcal·mol-1) and salt bridge (～3.66 kcal·mol-1).(ⅴ) Contribution of halide motifs to protein stability. Halide anions are traditionally recognized as the structure maker and breaker of water to indirectly influence the physicochemical and biological properties of biomacromolecules immersing in electrolyte solution, but here we are more interested in whether they can be structured in protein interior, forming that we named halide motifs, to stabilize the protein architecture through direct noncovalent interactions with their context? In the current work, we present a systematical investigation on the energy components in 782 high-quality protein halide motifs retrieved from the Protein Data Bank (PDB), by means of the continuum electrostatic analysis coupled with non-electrostatic considerations as well as hybrid QM/MM examination. We find that most halide motifs (91.6%) in our dataset are substantially stabilizing and their average stabilization energy is significantly larger than that previously obtained for sophisticated protein salt bridges (-15.16 vs.-3.66 kcal·mol-1). Strikingly, non-electrostatic factors, especially the dispersion potential, rather than the electrostatic aspect, dominate the energetic profile of the pronouncedly charged halide motifs, since the expensive cost for electrostatic desolvation penalty requires to be paid off using the income receiving from the favorable Coulomb interactions during the motif formation. In addition, all the energy terms involved in halide motifs, regardless of their electrostatic or non-electrostatic nature, show to highly depend on the degree of motif’s burial in protein, and the buried halide motifs are generally associated with a high stability.(vi) 2D depiction of interfacial NBMs for protein complexes. A program called the 2D-GraLab is described for automatically generating schematic representation of diverse NBMs across the protein binding interfaces. The input file of this program takes the standard PDB format, and the outputs are two-dimensional PostScript diagrams giving intuitive and informative description of the protein-protein interactions and their energetics properties, including hydrogen bond, salt bridge, van der Waals interaction, hydrophobic contact, p-p stacking, disulfide bond, desolvation effect, and loss of conformational entropy. To ensure the accuracy and reliability of determined interaction information, methods and standalone programs employed in the 2D-GraLab are all widely used in the chemistry and biology communities. The generated diagrams allow intuitive visualization of the interaction mode and binding specificity between two subunits in protein complexes, and by providing information on nonbonding energetics and geometrical characteristics, the program offers the possibility of comparing different protein binding profiles in a detailed, objective, and quantitative manner.更多还原