一种核糖核酸酶构象稳定性的研究

Aspergillus niger SA-13-20核糖核酸酶是从突变株A.niger SA-13-20分泌的胞外酶中分离出的一种新的核糖核酸酶。采用紫外光谱、荧光光谱和红外光谱研究了A.niger SA-13-20核糖核酸酶在不同pH值条件下的构象稳定性。紫外光谱和荧光光谱结果均表明该酶蛋白在酸性和弱碱性pH值下构象较稳定,当pH值高于9.6时构象不稳定;红外光谱结合去卷积和曲线拟合技术对蛋白质酰胺Ⅰ带的测定和处理结果表明,在室温下,pH 5.0时该酶蛋白二级结构中α-螺旋、β-折叠、转角和无规结构所占的成分分别为13.28%、42.30%、26.48%和17.95%。测得该酶的热解链温度Tm、解链熵变ΔSm及解链焓变ΔHm分别为70.1℃、644 J.mol-1.K-1及22.1 kJ.mol-1,表明该酶属于耐热能力较强的核糖核酸酶。研究结果有助于揭示该酶结构与功能的关系,推动其在科研和生产等方面的应用。     

中心蛋白C端半分子与Sm3+作用的光谱研究

在0.01 mol.L-1Hepes,0.15 mol.L-1NaCl,pH 7.4及室温条件下,通过荧光光谱、圆二色光谱和紫外差光谱的方法研究了Sm3+与八肋游仆虫中心蛋白C端半分子(C-terminal domain of Euplotes octocarinatus centrin,C-EoCen)III,IV结合位点的结合能力及结合后对蛋白质构象的影响。结果表明,Sm3+与C-EoCen结合后,蛋白质从关闭式构象转变为开放式构象,蛋白的疏水性结构域外露程度增强,同时蛋白的α-螺旋含量明显增大;Sm3+与C-EoCen的III,IV结合位点的条件稳定常数分别为:lgKIII=6.23±0.39,lgKIV=6.81±0.51。     

荧光光谱结合表面增强拉曼光谱法研究紫檀芪与人血清白蛋白相互作用

在模拟人体生理条件下,应用荧光光谱和表面增强拉曼光谱法研究了紫檀芪(PTE)与人血清白蛋白(HSA)之间相互作用机制.结果表明,HSA的荧光能被PTE静态猝灭,并伴随有非辐射能量转移作用,两者形成了1:1复合物,结合距离r=1.495 nm,结合常数KA=1.12×104(298 K)、4.07×104(304 K)和2.45×105 L/mol(310 K).表面增强拉曼光谱研究揭示,PTE分子通过甲氧基与HSA进行结合;热力学数据表明,二者间的作用主要为疏水作用;标记竞争实验指出PTE优先结合HSA的位点Ⅲ.三维荧光光谱、同步荧光光谱和表面增强拉曼光谱结果显示,与PTE作用后,HSA构象发生变化,导致色氨酸残基周围环境疏水性降低,但对PTE分子构象影响不大.     

光谱法研究Cu2+与肌红蛋白的相互作用

用紫外吸收光谱、荧光光谱、同步荧光光谱及圆二色(CD)谱研究了Cu2+与肌红蛋白(Mb)的相互作用. 结果发现, Cu2+使Mb的紫外吸收增强, 峰位蓝移, 说明Cu2+与Mb发生了较强的相互作用; Mb的特征荧光峰猝灭, 且随着温度升高猝灭常数Ksv降低, 表明Cu2+对Mb的荧光猝灭机制属于静态猝灭; 计算了不同温度下的结合常数和结合位点数; 由van′t Hoff方程计算出ΔH和ΔS分别为-11.60 kJ/mol和33.77 J·(mol·K)-1, 得出二者之间的作用力主要为静电力; 并依据Förster非辐射能量转移理论确定了给体-受体间的结合距离r=2.56 nm. 同步荧光光谱表明, Cu2+对Mb的构象产生影响, 使色氨酸残基的疏水性下降. CD光谱测得加入Cu2+后, 二级结构发生改变, 使α-螺旋含量降低.     

β-转角肽的溶液构象

主要报导TEM-1β-内酰胺酶的天然蛋白类抑制剂BLIP中一段多肽B1的溶液构象研究 结果.在磷酸盐缓冲溶液中,通过圆二色光谱、傅立叶红外光谱和核磁共振谱研究了B1的二 级结构特征.实验结果表明,B1在溶液中形成了β-转角结构,为在溶液中单独研究β-转 角结构形成与稳定性提供了良好的模板.β-转角在溶液中可以独立存在,表明β-转角在 蛋白质折叠过程中可能具有重要作用.     

铱(IV)离子与人血丙种球蛋白的作用研究

在0.1 mol•L^－1醋酸-醋酸钠(pH 5.0)体系中, 采用紫外吸收光谱、荧光光谱及同步荧光光谱法研究了人血丙种球蛋白(gamma seroglobulinum humanum, 简称GSH)与铱(IV)离子的相互作用. 结果表明, Ir(IV)离子使人血丙种球蛋白的构象发生了改变, α-螺旋含量减少, 并且用同步荧光光谱发现Ir(IV)离子与人血丙种球蛋白的作用位点更接近于色氨酸, 从而使色氨酸残基的疏水性略有减小. 荧光光谱结果表明Ir(IV)对人血丙种球蛋白内源荧光(342 nm)产生了较强的荧光猝灭作用, 根据不同温度下Ir(IV)对人血丙种球蛋白的荧光猝灭作用, 证明了这种荧光猝灭为静态猝灭机制, 计算了其结合常数和结合位点数, 从而得出了静电作用力为其主要的作用力.     

胰高血糖素样肽-1受体胞外区域突变体复合物的动力学研究

采用分子动力学模拟方法研究了胰高血糖素样肽-1(GLP-1)与GLP-1受体(GLP-1R)胞外区域的相互作用.结果表明,配体的结合导致受体的构象发生改变,Loop2区域的氨基酸Pro90和Trp91以及C末端的Glu128向配体移动.根据保守位点突变受体(P73A,V81L,Y88A,P90A和W91A)后所得多肽模拟数据,发现Loop2区域在突变体中的结构和柔性均发生了明显变化,Trp91和Tyr88的突变将导致配体亲和力丧失.研究结果证明,P73A突变型受体和野生型受体分别与配体相互作用后,二者数值差别不大,因此Pro73不是关键残基;V81L突变体则会导致配体亲和力的丧失.该结果为GLP-1药物设计提供了重要理论依据.     

三链DNA |dA10 2DT10的近红外付立叶拉曼光谱

最近通过核磁共振、振动光谱和分子力学模拟研究表明,在溶液中三螺旋DNA的构象具有一定程度的复杂性和多样性．本文首次采用近红外付立叶拉曼光谱研究了三螺旋DNA dA10•2d10和相应的双螺旋DNA dA10•dT10在溶液中的构象．结果表明该三螺旋DNA同时存在C3’－内褶／反对称（A－型）和C2’－内褶／反对称（B－型）两种构象,而且在823cm－1处的较强谱带暗示还存在一种处于A和B构型之间的中间构型．对出现在1218cm-1和638cm-1处、归属于对糖环构象敏感的胸腺嘧啶残基的两个特征谱带进行分析,获得了除C2’－内褶构象外其它构型存在的证据．另外,双螺旋DNA的FT－Raman光谱表明该双螺旋DNA在溶液中以A－和B－两种构型共存．     

铱(IV)离子与人血丙种球蛋白的作用研究

在 0.1 mol?L-1醋酸-醋酸钠(pH 5.0)体系中, 采用紫外吸收光谱、荧光光谱及同步荧光光谱法研究了人血丙种球蛋白(gamma seroglobulinum humanum, 简称 GSH)与铱(IV)离子的相互作用. 结果表明, Ir(IV)离子使人血丙种球蛋白的构象发生了改变, α-螺旋含量减少, 并且用同步荧光光谱发现 Ir(IV)离子与人血丙种球蛋白的作用位点更接近于色氨酸,从而使色氨酸残基的疏水性略有减小. 荧光光谱结果表明Ir(IV)对人血丙种球蛋白内源荧光(342 nm)产生了较强的荧光猝灭作用, 根据不同温度下 Ir(IV)对人血丙种球蛋白的荧光猝灭作用, 证明了这种荧光猝灭为静态猝灭机制, 计算了其结合常数和结合位点数, 从而得出了静电作用力为其主要的作用力.     

氯硝柳胺及其衍生物与钥孔戚血蓝蛋白的相互作用

以氯硝柳胺为原料合成了聚乙二醇200基氯硝柳胺. 采用荧光光谱、同步荧光光谱、紫外吸收光谱和圆二色谱研究了氯硝柳胺及其衍生物与钥孔戚血蓝蛋白(KLH)的相互作用. 结果表明, 两种药物分子对KLH的荧光猝灭机制属于静态猝灭; 由Lineweaver-Burk方程计算出不同温度下结合常数K, 但是聚乙二醇200基氯硝柳胺与KLH的作用相对较弱; 由Van′t Hoff方程计算出ΔH和ΔS平均值, 结合力主要为静电作用力; 热力学函数计算结果表明, 氯硝柳胺及其衍生物与KLH的作用过程是一个熵增加、Gibbs自由能降低的自发分子间作用过程; 根据Förster非辐射能量转移机制求得给体与受体间的结合距离r均小于7 nm; 同步荧光光谱表明, 氯硝柳胺及其衍生物能够被血蓝蛋白存储和转运, 但结合时对蛋白构象有一定影响; 圆二色谱测得加入两种药物后, KLH的α-螺旋含量均降低, 二级结构发生改变. 通过比较氯硝柳胺及其衍生物与KLH的相互作用, 初步探讨了分子结构与其结合能力之间的联系.     

Intramolecularly lactam stapled oxyntomodulin analogues inhibit cancer cell proliferation in vitro

As a glucagon (GCG) receptor (GCGR) and glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) dual agonist, oxyntomodulin (OXM) has been attracting scientific attentions due to its efficacies of suppressing appetite, increasing energy expenditure, and inducing body weight loss in obese humans. Based on the scaffold of native OXM, specific helix-favoring amino acids substitutions and the consequent salt bridge formations were believed to offer enhanced and balanced GCGR/GLP-1R activations through increasing α-helical conformation. Novel OXM analogues are obtained by intramolecular lactam stapling of positions [Glu16 & Lys20] or [Lys17 & Glu21] to further strengthen conformationally constrained stabilization. Even though the lactam staple does not provide additional dual GCGR/GLP-1R activations in vitro, the stapled OXM analogues are firstly reported to have higher or lower anti-PANC-1 cell proliferation activity, meanwhile which has no obvious inhibitory effect on the proliferation of HeLa cells. Therefore, it is speculated that the stapled analogues may have the potential to inhibit the proliferation of specific cancer cell types. Among the stapled peptides as well as their precursors, analogue 6 has the most prominent anti-PANC-1 proliferation activity with the IC₅₀ value of 115.1 μmol/L. Its mechanism of actions including effective signal pathways should be worth further investigations in future.     

pH值对丝素蛋白构象转变的影响

模仿家蚕吐丝过程中伴随丝素蛋白自然脱水的纤维化过程,研究了再生丝素蛋白在各种pH值的磷酸盐缓冲溶液体系中自然干燥脱水成膜后的构象转变.利用激光拉曼散射光谱及其二维相关光谱,定性分析了丝素蛋白酰胺区(1600～1700cm^-1)散射峰的相关组成及结构.在此基础上,利用¹³CCP-MAS固体核磁共振谱对丝素蛋白丙氨酸C_β峰(δ14.5～22)进行了解析拟合.从而确定了体系中与Silk及Silk构象相关的组成含量与pH值的关系.结果表明,pH=5.2的酸性溶液有利于蚕丝丝素蛋白从Silk向Silk构象转变,而中性与碱性溶液(pH=6.9和8.0)则对丝素蛋白的构象转变影响甚小.     

Separation of diadenosine polyphosphates by capillary electrophoresis

The influence of buffer composition and pH on the electrophoretic behavior of diadenosine polyphosphates with a phosphate chain ranging from two to five phosphate groups has been examined. The electrophoretic mobility in carbonate buffer increases according to the number of phosphates, whereas in borate buffer the mobility changes in an irregular way as a function of pH. This finding can be rationalized by a well-known interaction of borate with ribose rings, which modifies the charge and the hydrodynamic radius of each diadenosine polyphosphate in a different way. Our study shows that the best separation of diadenosine polyphosphates can be achieved at the highest pH values of the range examined both in borate and carbonate buffers.     

植物白头翁毒蛋白的分离、纯化及其组分测定

植物白头翁（ａｍｅｎｏｎｅ）茎的抽提液经ＣＭ－ＳＦＦ柱和ＳｅｐｈａｃｒｙｌＳ－２００柱分离纯化,得到一种毒蛋白,用高效凝胶蛋白柱和反相高效液相色谱法结合光电二极管阵列检测器确认分离峰的纯度,在高效凝胶蛋白柱上制备了少量毒蛋白纯样,测定了蛋白分子量和氨基酸组成。     

Study on the supramolecular systems of 5-(2-hydroxy phenyl)-10,15,20-tris (4-methoxy phenyl) porphyrin with cyclodextrins

In neutral phosphate buffer solutions of pH 7.4, the inclusive complexation of 5-(2-hydroxy phenyl)-10,15,20-tris(4-methoxy phenyl) porphyrin (o-HTPP) with alpha-cyclodextrin (alpha-CD), beta-CD, heptakis (2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD), SBE-beta-CD, HP-beta-CD and gamma-CD has been examined by means of UV-vis and fluorescence spectroscopy. The formation of inclusion complexes has been confirmed on the base of changes of spectroscopy properties. The o-HTPP forms 1:2 inclusion complexes with TM-beta-CD and 1:1 inclusion complexes with the other five cyclodextrins. The formation constants (K) of o-HTPP for the formation of the inclusion complexes have been estimated from the absorbance and fluorescence intensity changes in neutral phosphate buffer solutions. The K value (2.89x10(7)), which is the formation constant for the formation of the 1:2 inclusion supramolecular, is nearly 10(4) times than those of the 1:1 inclusion complexes. Compared to the other five cyclodextrins, the strongest inclusion ability of TM-beta-CD can be explained that the hydrogen bond plays significant role in the inclusion process. UV-vis experiments also showed that the cavity of TM-beta-CD causes the transform of the state of o-HTPP. In addition (1)H NMR data and 2D-ROSEY NMR spectra support the inclusion conformation of the o-HTPP-CD supramolecular system, indicating the interaction mechanism of inclusion processes.     

Slow proton transfer from the hydrogen-labelled carboxylic acid side chain (Glu-165) of triosephosphate isomerase to imidazole buffer in D2O

The catalytic base at the active site of triosephosphate isomerase (TIM) was labelled with -H by abstraction of a proton from substrate d-glyceraldehyde 3-phosphate to form an enzyme-bound enediol(ate) in D2O solvent. The partitioning of this labelled enzyme between intramolecular transfer of -H to form dihydroxyacetone phosphate (DHAP), and irreversible exchange with -D from solvent was examined by determining the yields of H- and D-labelled products by 1H NMR spectroscopy. The yield of hydrogen-labelled product DHAP remains constant as the concentration of the basic form of imidazole buffer is increased from 0.014 to 0.56 M. This shows that the active site of free TIM, which has an open conformation needed to allow substrate binding, adopts a closed conformation at the enediolate-complex intermediate where the catalytic side chain is sequestered from interaction with imidazole dissolved in D2O.     

Effect of buffer on heparin binding and sensing in competitive aqueous media

Abstract

Although buffer-specific effects on molecular recognition are known in biological science, they remain rare in supramolecular chemistry. The binding between a cationic dye, mallard blue (MalB) and polyanionic heparin in aqueous NaCl (150 mM) is studied in three commonly used buffers (Tris-HCl, HEPES, Phosphate, each 10 mM). Although MalB has a very similar UV–visible spectrum in each buffer, the sensory response towards heparin was different in each case. This can be ascribed to differences in the complex formed. In Tris-HCl which has the least competitive chloride counter-anions, MalB exhibits a hypsochromic shift of 25 nm, assigned to strong binding and aggregation of the dye on heparin. In more competitive HEPES, containing a sulfonate anion, there is weaker binding and less aggregation of MalB along the heparin; the hypsochromic shift is only 15 nm. In phosphate buffer, MalB can interact quite strongly with buffer phosphate anions; although heparin binding is still observed, the hypsochromic shift associated with dye aggregation is only 5 nm. As such, specific buffer interactions with the MalB–heparin complex mediate host–guest binding and sensing. Buffer choice must be made carefully in studies of molecular recognition – we would caution against using phosphate and sulfonate containing buffers when studying electrostatic binding.     

Effect of pH and small inorganic ions on binding of sulfadimethoxine and sulfaphenazole to human serum albumin measured by circular dichroism

The binding of sulfadimethoxine and sulfaphenazole to human serum albumin (HSA) has been shown by circular dichroism measurements to be dependent on the N-B transition. The secondary drug binding sites were found to be optically active in the B conformation form in HSA but optically inactive in the N form. Moreover, the drug-HSA interaction in Tris-HCl buffer seems to be more sensitive to the conformational change in HSA, compared with that in the phosphate buffer.     

Study on the Conformation of Cyclodecapeptide Loloatin C with Obvious Antibiotic Activity

The conformation of cyclodecapeptide loloatin C with obvious antibiotic activity has been investigated in 2,2,2-trifluoroethanol/sodium acetate buffer solution and then characterized by Fr-IR, CD and NMR spectrum. The results of FT-IR show that there exists β-strand or β-tum secondary structure in the molecule. According to the CD spectrum, the helical turn is dominant but the β-turn structure also exists. Conformation of the whole molecule is probably a helical β-turn.The chemical shifts and coupling constants prove the existence of a β-structure in the regions of Val,Orn2 and Leu3. NOESY data and temperature gradients of amide protons suggest that the molecular conformation is a dumbbell-like structure with the waist located between ornithyl (position 2) and D-phenylalanyl (position 7) and β-turn on both ends.     

Glucagon-like peptide 2 (GLP-2), an intestinotrophic mediator

Glucagon-like peptide 2 (GLP-2) is a newly discovered gastrointestinal peptide with 33% sequence homology to glucagon. GLP-2 has attracted interest because of its potent intestinotrophic endocrine/paracrine actions. The peptide, consisting of 33-amino-acid, results from expression of the glucagon gene in the enteroendocrine L-cells of the intestinal mucosa, from where it is released mainly in response to luminal contact with unabsorbed nutrients. In addition to mucosal growth, GLP-2 enhances activities of several intestinal brush-border enzymes, and it delays gastric transit, thereby increasing the intestinal capacity for nutrient absorption. Thus, it appears that GLP-2 serves to ensure an optimal intestinal capacity. The physiological responses following exogenous administration of GLP-2 have been intensely investigated, and these appear to be rather specific for the gut, which is concordant with the localization of the GLP-2 receptor. In addition, treatment with GLP-2 in experimental animal models of several enteropathies indicates that GLP-2 ameliorates most of the observed intestinal abnormalities in these conditions. Following secretion to the blood stream, the intact peptide is degraded rather rapidly by an aminopeptidase. To circumvent the rapid and widespread metabolization of intact GLP-2, degradation-resistant synthetic GLP-2 analogues have been developed together with other approaches, such as inhibition of the GLP-2 degrading enzyme. This is of particular interest with respect to developing GLP-2 into a useful therapeutic agent in conditions with compromised intestinal function, since the first clinical trial has already indicated the potential of GLP-2 treatment in patients with short bowel syndrome.