可溶性鸟苷酸环化酶H-NOX结构域一氧化碳结合态的结构研究

发布时间：2018-01-01 01:13

本文关键词：可溶性鸟苷酸环化酶H-NOX结构域一氧化碳结合态的结构研究　出处：《吉林大学》2016年博士论文　论文类型：学位论文

【摘要】：可溶性鸟苷酸环化酶(soluble Guanylate Cyclase,s GC)是一种含血红素的卟啉蛋白,在高等生物体内的NO-c GMP信号转导通路中催化GTP生成第二信使c GMP,调节心血管系统、神经系统以及细胞的凋亡等生命代谢活动。s GC由一对异源二聚体组成,分别为不含卟啉的α亚基和含卟啉的β亚基,其中β亚基N端的194个氨基酸(s GCβ1_(1-194)H-NOX)是已知的能够稳定结合卟啉的最小片段。作为NO受体酶,s GC对气体配基的结合性质有着重要的意义。一直以来,s GC的全酶只能从动物脏器、组织中提取,耗时很长而且提纯量很少。随着技术的发展,来源于细菌具有高度保守序列和s GC高同源性的H-NOX家族蛋白的发现,为表达s GC奠定了基础,选择适合的宿主和载体可以实现H-NOX家族蛋白和s GCβ1亚基片段的表达。Ns H-NOX是目前发现的H-NOX家族蛋白中与s GC同源性最高的一类蛋白和s GCβ1_(1-194)H-NOX一起因与s GC高度一致的序列组成和相似的气体配位性质成为了研究s GC结构与功能的新焦点。在本论文的研究中,我们构建了s GCβ1_(1-194)H-NOX(p ET-20b+s GCβ1_(1-194))和Ns H-NOX(p UC19+anaerobic promoter+hnox)的原核表达体系,用大肠杆菌作为宿主表达得到了纯度较高的s GCβ1_(1-194)H-NOX和Ns H-NOX。Ns H-NOX和s GCβ1_(1-194)H-NOX与s GC全酶有着极其相似的气体结合性质。为了研究H-NOX结构域的一氧化碳(CO)结合性质以及CO结合态蛋白的结构变化,我们分别通过共振拉曼光谱(r RS)和分子动力学模拟(MD)以及密度泛函理论计算(DFT)的方法来探讨这一问题。在对Ns H-NOX和s GCβ1_(1-194)H-NOX的共振拉曼光谱研究中我们发现两种蛋白都存在类似于s GC全酶的两种不同的Fe-CO伸缩振动模式(v Fe-CO),相对应的存在两种不同的CO结合态也就是存在两种不同的CO配位结构。通过对不同长度的s GCβ1片段和s GC全酶以及我们的样品进行高斯拟合分峰处理,Ns H-NOX的v Fe-CO出现在494cm-1和470cm-1,而s GCβ1_(1-194)H-NOX则出现在489cm-1和473cm-1且两种组分的相对比例在不同片段长度的s GCβ1亚基中是不同的,进一步说明了每一种v Fe-CO振动模式对应着一种不同的CO结合态结构,而且在不同的蛋白中这两种不同结构所占的比例不同。在前期工作中,利用Ns H-NOX晶体结构为模板模建了s GCβ1 H-NOX的3D模型,本研究中对s GCβ1 H-NOX和Ns H-NOX在CO结合时的结构变化和400ns内的分子动力学轨迹进行了讨论。150ns后,在s GCβ1 H-NOX中,水分子在CO附近大量聚集,而在Ns H-NOX中,水分子仅有少量分布。在s GCβ1H-NOX中,由于αF螺旋的112位酪氨酸(Y112)和αD螺旋与αE螺旋之间的83位酪氨酸(Y83)能够形成氢键网络,使αF螺旋发生扭曲,导致其上方血红素卟啉大环发生位移,形成了可以容纳水分子进出的水通道,水分子通过这个通道进入到了血红素远端口袋,与轴向的CO之间形成了氢键网络。而Ns H-NOX中112位是苯丙氨酸(F112)不能形成氢键网络,没有水分子可以进入的通道,CO附近不存在长时间停留的水分子,但是仍然有极少部分水分子可以以其他的方式进入远端口袋内部,与CO之间形成部分氢键。s GCβ1_(1-194)H-NOX的远端血红素口袋更多的暴露在溶剂中,以及水分子迁移通道的存在,导致s GCβ1_(1-194)H-NOX与Ns H-NOX相比有更快的氧化速率。为了验证水分子进入到血红素远端口袋影响了CO结合态的结构的推论,我们通过DFT量化计算的方法对(Im H)Fe P(CO)体系在水分子存在条件下的拉曼光谱进行了计算,计算结果与共振拉曼实验结果一致。我们讨论了几种具有不同极性的氢键供体H2S,NH3,H2O,HF对氢键形成与否与不同氢键强度下(Im H)Fe P(CO)体系的轨道能级分布、电荷密度分布。在水分子氢键存在的体系中,卟啉大环平面上四个N原子周围电荷密度减小,中心Fe原子电荷密度增加,而在卟啉轴向上,Fe原子和C原子之间电荷密度明显增大,说明C原子和Fe原子之间的相互作用增强,反映在拉曼光谱上为v Fe-CO的频率增大,Fe-CO之间形成反馈键,Fe的dπ电子进入CO的π*轨道,降低了CO内部成键强度,随着氢键的增强,C-O之间的电荷密度明显的降低,表明C原子和O原子之间的作用变弱,v C-O振动频率变小。H-NOX结构域Fe-CO的伸缩振动v Fe-CO出现在两个不同位置的原因在于存在能够进入到血红素远端口袋中的水分子与配位的CO的O形成氢键,从而改变了Fe-CO的振动模式,同时也代表着两种不同CO结合态结构的存在。即在较高波数的为形成氢键的Fe-CO…HOH的振动模式,而较低波数的是Fe-CO不形成氢键的振动模式。我们对s GCβ1 H-NOX和Ns H-NOX的CO结合态的结构研究,对于进一步阐明可溶性鸟苷酸环化酶s GC的激活机制、s GC和H-NOX家族蛋白的结构功能以及相关药物的研发具有重要的意义。
[Abstract]:Soluble guanylate cyclase (soluble Guanylate Cyclase, s GC) is a heme porphyrin protein catalyzed by GTP NO-c GMP signal transduction pathway in higher organisms in the formation of the second messenger C GMP, regulating the cardiovascular system, nervous system and cell apoptosis of life and metabolic activity of.S GC by a heterologous two dimers, respectively, alpha subunit containing porphyrin and porphyrin containing beta subunit of 194 amino acids including beta subunit N end (s GC beta 1_ (1-194) H-NOX) is known to be stable with minimum porphyrin fragment. As NO receptor enzyme, has an important significance of the combination GC on the properties of s gas ligands. Since the s GC holoenzyme only from animal organs, tissues, are time-consuming and purification of few amount. With the development of technology, from bacteria to highly conserved s and GC high homology of H-NOX family proteins found for the table Laid the foundation of s GC, select the appropriate host and vector can achieve H-NOX protein family and s GC beta 1 subunit fragment of.Ns H-NOX expression is found in the H-NOX protein family in s GC with the highest homology of proteins and s GC beta 1_ (1-194) H-NOX and s GC sequence with gas highly consistent composition and similar coordination properties has become a new focus of research on the structure and function of s GC. In this paper, we constructed the s GC beta 1_ (1-194) H-NOX (P ET-20b+s GC 1_ (1-194)) and Ns H-NOX (P UC19+anaerobic promoter+hnox) prokaryotic expression system. Using Escherichia coli as the host expression of the high purity of the s GC beta 1_ (1-194) H-NOX and Ns H-NOX.Ns H-NOX and s GC beta 1_ (1-194) H-NOX and s GC enzyme has a similar binding properties of the gas. In order to study the H-NOX domain of carbon monoxide (CO) and CO binding properties of bound protein the Structural changes, we respectively through resonance Raman spectroscopy (R RS) and molecular dynamics simulation (MD) calculation and density functional theory (DFT) method to study the problem. In the Ns H-NOX and s GC 1_ (1-194) resonance Raman spectroscopy in the study of H-NOX we found that two proteins are two different Fe-CO stretching mode is similar to the s GC holoenzyme (V Fe-CO), corresponding to two different CO bound is also there are two kinds of CO coordination structure. According to the different length of the s beta 1 GC fragment and s GC holoenzyme and our sample of Gauss fitting peak processing, Ns H-NOX V Fe-CO in 494cm-1 and 470cm-1, and s GC beta 1_ (1-194) is different from that of H-NOX appears in the relative proportion of 489cm-1 and 473cm-1 and two kinds of components in different fragment length s GC beta 1 subunit, further explains each V Fe-CO vibration mode a corresponding Different CO bound structure, and in different protein structure of the two different proportion. In previous work, the 3D model using Ns H-NOX s GC beta crystal structure built as template model for 1 H-NOX, the study of s GC H-NOX and Ns H-NOX in beta 1 CO binding when the structure of molecular dynamics trajectory change and 400ns were discussed after.150ns in the s GC beta 1 H-NOX, water molecular aggregation in the vicinity of the CO large, while in Ns H-NOX, only a small amount of the distribution of water molecules. In the s GC beta 1H-NOX, the tyrosine 112 alpha helix F (Y112) 83 between tyrosine and alpha D alpha E spiral and spiral (Y83) can form a hydrogen bond network, the alpha F spiral twist, lead to the above the heme macrocycle displacement, the formation of water channel can contain water molecules and water molecules through the channel into the heme distal pocket, and axial CO Between the formation of a hydrogen bond network. While the Ns H-NOX 112 (F112) is a phenylalanine cannot form hydrogen bond network, no water molecules can enter the channel, CO does not exist near the water molecule stays for a long time, but there are still a few water molecules can enter the distal end of internal bag in other ways, part of the hydrogen.S GC beta the formation of 1_ and CO (1-194) H-NOX of the distal heme pocket more exposed to the solvent, and the existence of water molecule migration channel, resulting in s GC beta 1_ (1-194) H-NOX and Ns H-NOX have faster oxidation rate compared. In order to verify the water into the heme distal pocket of CO combined with corollary state structure the method we calculated by DFT (Im H) to quantify the Fe P (CO) system was calculated in the presence of water molecules under the conditions of Raman spectra, the calculated results agree with experimental results. The resonance Raman we discussed several Different polar hydrogen bond donor H2S, NH3, H2O, HF on the formation of hydrogen bonds and hydrogen bond strength under different (Im H) Fe P (CO) orbital distribution system, the distribution of charge density. In the presence of hydrogen bonded water molecules of porphyrin macrocycle plane four N atomic charge density around. Fe, atomic charge density increased, and in axial, between Fe and C atoms in the charge density increased significantly, indicating a stronger interaction between C and Fe atoms, reflected in the Raman spectra for V Fe-CO frequency increases feedback bond formation between Fe-CO and Fe D CO into the pi pi electron * track, reduce the CO internal bond strength, with hydrogen bonds increased, the charge density between C-O decreased obviously and that between C and O atoms becomes weaker, V Fe-CO V C-O stretching vibration frequency of small.H-NOX domain Fe-CO appeared in two different positions of reason It is the existence of water molecules that can enter into the heme distal pocket and form hydrogen bonds with the O of the coordinated CO, thus changing the vibrational mode of Fe-CO. It also represents the existence of two different CO binding states. That is, the Fe-CO with hydrogen bonds at higher wavenumber. The vibration modes of HOH, while the lower wavenumber is vibration mode Fe-CO cannot form hydrogen bond. Research on structure of S combined with our GC beta 1 H-NOX and Ns H-NOX CO, to further elucidate the mechanisms of activation of soluble guanylate cyclase s GC, plays an important role in structure and function of s GC and H-NOX development protein family and related drugs.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q55

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