脲和盐酸胍诱导的卵清溶菌酶分子去折叠过程中各稳定构象态的分布和过渡

以内源荧光光谱和荧光相图法研究了脲和盐酸胍诱导的卵清溶菌酶分子的去折叠过程,结果表明,当变性液中脲和盐酸胍的浓度分别约为4.0和3.0 mol/L时,卵清溶菌酶分子的去折叠过程均存在一个折叠中间态,这两个去折叠过程均符合"三态模型".在卵清溶菌酶分子"三态"去折叠过程的基础上,通过变性剂分子和卵清溶菌酶分子之间的缔合一...     

盐酸胍诱导的变性卵清溶菌酶分子重折叠过程及其各稳定构象态的分布和过渡

采用变性和非变性电泳、 高效凝胶排阻色谱、 内源荧光发射光谱和荧光相图以及生物活性测定等方法, 研究了盐酸胍诱导的变性卵清溶菌酶分子的重折叠过程及此过程中卵清溶菌酶分子各稳定构象态的分布和过渡. 结果表明, 当复性液中盐酸胍浓度分别约为5.0和2.4 mol/L时, 变性卵清溶菌酶分子的重折叠过程各存在1个稳定折叠中间态, 重折叠过程符合"四态模型". 在卵清溶菌酶分子四态重折叠过程基础上, 结合盐酸胍与卵清溶菌酶分子之间的缔合-解离平衡, 给出了一个定量描述变性剂诱导的蛋白质分子复性过程中蛋白质分子复性率随溶液中变性剂浓度变化的方程. 该方程包含2个特征折叠参数, 一个是蛋白质分子从一个稳定构象态过渡到另一个稳定构象态的热力学过渡平衡常数k; 另一个是在此过程中平均每个蛋白质分子所结合的变性剂分子数目m. 通过这2个特征折叠参数能够定量描述盐酸胍诱导的变性卵清溶菌酶完全去折叠态、 折叠中间态和天然态分子随复性液中盐酸胍浓度变化的分布和过渡情况.     

脲和盐酸胍诱导溶菌酶去折叠的荧光相图法研究

用荧光相图法分别研究了脲和盐酸胍诱导卵清溶菌酶去抓叠的过程。当变性体 系中无还原剂2－巯基乙醇存在、脲浓度从0变化至4.0 mol/L（或盐酸胍浓度从0变 化至3.0 mol/L）时,溶菌酶从天然态转变为部分折叠中间态,当脲浓度从4.0 mol/L变化至8.0 mol/L（或盐酸胍浓度从3.0 mol/L变化至6.0 mol/L）时,溶菌 酶从中间态转变为去折叠态,此时该蛋白的变性过程符合“三态模型”。而当变性 体系中有该还原剂存在时,溶菌酶则由天然态直接转变为去折叠态,此时脲诱导该 蛋白去折叠的过程符合曲型的“二态模型”。实难结果表明荧光相图法可以检测蛋 白南去抓叠的中间态。     

血红蛋白和细胞色素c构象去折叠行为研究

利用紫外-可见吸收和荧光光谱法研究了血红蛋白(Hb)与细胞色素c(Cytc)两种血红素蛋白的去折叠行为。采用化学变性剂盐酸胍(GdHCl)和尿素(Urea)诱导两种蛋白构象去折叠,阐述了两种蛋白的去折叠机理。Hb的血红素(Heme)辅基通过与卟啉铁原子和组氨酸配位,与肽链键合的稳定性较差,在3.0 mol/L的盐酸胍作用下即发生解离。而Cyt c的Heme辅基通过卟啉与半胱氨酸形成二硫键呈现较强的稳定性,盐酸胍浓度达到6.0 mol/L也难使其发生解离。该研究为阐释蛋白构象与功能之间的关系提供了重要依据。     

脲和盐酸胍诱导过氧化氢酶去折叠的研究

用荧光相图法分别研究了脲和盐酸胍诱导牛肝过氧化氢酶去折叠的过程。当脲 浓度从0依次增大至0.50,4.5和8.0 mol/L时,过氧化氢酶从天然四聚体依次转变 为蓬松的四聚体、部分折叠的无活性二聚体和去折叠态,而当盐酸胍浓度从0依次 变化至0.65,2.5和6.0 mol/L时,过氧化氢酶则从天然四聚体集资转变为部分折叠 的激活二聚体、部分折叠的单体和去折叠态,这表明无论是用脲还是用盐酸胍作为 变性剂,该蛋白的变性过程都符合“四态模型”,但这两种变性剂诱导该蛋白去折 叠的途径和机制有较大差异。实验结果表明荧光相图法可以检测蛋白质去折叠的中 间态。用等温滴定量去热法研究了盐酸胍诱导过氧化氢酶去折叠过程的热力学, 25.0 ℃时低浓度盐酸胍诱导该蛋白从天然四聚体转变为部分折叠的激活二聚体的 本征摩尔构象变化焓、Gibbs自由能和熵分别为-69.2 kJ·mol~(-1),6.43 kJ· mol~(-1)和-254 J·K~(-1)·mol~(-1),据此推断盐酸胍通过熵效应和静电效应来 稳定和激活该二聚体。     

盐酸胍诱导的淀粉液化芽孢杆菌α-淀粉酶去折叠过程的研究

分别用内源荧光光谱法、荧光相图法、荧光探针法、荧光猝灭法、蛋白质电泳法以及体积排阻色谱法研究了盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程. 内源荧光光谱和荧光相图结果表明, 当变性液中盐酸胍浓度约为1.0 mol/L时, 芽孢杆菌a-淀粉酶的去折叠过程中出现一个部分折叠中间体, 其去折叠过程符合“三态模型”; 荧光探针结果表明, 在溶液中盐酸胍浓度约为1.0 mol/L时, 中间态芽孢杆菌a-淀粉酶分子中存在着能够与探针分子1-苯胺 基-8-萘磺酸(ANS)结合的稳定的疏水区域; 荧光猝灭研究给出了不同程度变性的淀粉液化芽孢杆菌a-淀粉酶中的Trp的分布情况, 结果表明中间态芽孢杆菌a-淀粉酶分子中能够被碘化钾猝灭的位于分子表面的色氨酸残基数目达到最大的8个; 蛋白电泳和体积排阻色谱结果表明, 在盐酸胍诱导的芽孢杆菌a-淀粉酶分子的整个去折叠过程中, 不会以共价键或非共价键形式形成芽孢杆菌a-淀粉酶分子之间的集聚体或集聚体沉淀. 在此基础上, 对盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程进行了描述.     

多方法组合分析脲诱导的淀粉液化芽孢杆菌α-淀粉酶去折叠和重折叠过程的构象转化

以变性和非变性电泳、体积排阻色谱、内源荧光发射光谱、荧光相图、荧光猝灭以及活性测定等组合分析方法,研究了脲诱导的淀粉液化芽孢杆菌α-淀粉酶分子的去折叠和重折叠过程。结果表明,在脲诱导的芽孢杆菌α-淀粉酶分子的去折叠和重折叠过程中,芽孢杆菌α-淀粉酶分子始终以单分子形式存在,不会形成分子间的聚集体或聚集体沉淀。当变性液或复性液中脲浓度约为4.0mol/L时,芽孢杆菌α-淀粉酶分子的去折叠和重折叠过程中均出现一个部分折叠中间体,两个过程均符合"三态模型"。脲诱导的芽孢杆菌α-淀粉酶分子重折叠过程的复性曲线几乎与芽孢杆菌α-淀粉酶分子去折叠过程的残余活性率曲线重合。通过这些结果,并结合盐酸胍诱导的芽孢杆菌α-淀粉酶分子的去折叠和重折叠过程,推断脲和盐酸胍诱导的芽孢杆菌α-淀粉酶分子的去折叠和重折叠过程分别是相互可逆的。     

含有二硫键的蛋白质在6mol/L盐酸胍中变性后不是完全无序的

本文用紫外差光谱、紫外二阶导数光谱和圆二色光谱,对二硫键完整和经还原并氨酰羧甲基化(以下简称还原)的核糖核酸酶A(RNase A),牛血清白蛋白(BSA)和溶菌酶在6 mol/L盐酸胍中变性后的结构进行了比较。尽管在圆二色光谱中,二硫键完整和还原的变性蛋白都看不到α-螺旋和β-折叠等有序二级结构的存在,但是,与二硫键还原的变性蛋白相比,含有完整天然二硫键的变性蛋白在芳香族氨基酸残基侧链的暴露程度上明显较低。这些结果说明含有完整天然二硫键的蛋白在6mol/L盐酸胍中变性后可能仍然保留有一定程度的有序空间结构。     

二硫键完整对蛋白质在盐酸胍中变性时伸展的影响——Fourier红外光谱研究

利用Fourier红外光谱对二硫键完整和还原的核糖核酸酶A(RNase A),牛血清白蛋白(BSA)和溶菌酶在6mol/L盐酸胍中变性后的结构进行了比较。这三种蛋白质,在二硫键完整和还原状态下变性后的Fourier红外酰胺Ⅰ带光谱都表现了不同程度的差异,但是三种还原蛋白质在完全变性后的光谱却非常相似。说明含有完整天然二硫键的蛋白在6mol/L盐酸胍中变性后仍然有相当程度的残留有序结构,而二硫键断开的蛋白质完全变性后在结构上却是相似的。     

盐酸胍/Ⅰ型胶原分散体系的流变性

采用胃蛋白酶降解法从猪皮中提取了胶原蛋白, 用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定为Ⅰ型胶原; 红外及紫外光谱表明胶原分子中存在三螺旋结构. 分别采用小幅振荡剪切法、 恒定剪切速率法及滞后环法研究了盐酸胍浓度及作用时间对Ⅰ型胶原蛋白体系流变性的影响. 研究表明, 随盐酸胍浓度的增大及作用时间的延长, 盐酸胍/胶原分散体系由假塑性流体逐渐接近于牛顿流体. 在所研究的盐酸胍浓度范围(0~6.0 mol/L)内, 盐酸胍/胶原分散体系的触变性类型随盐酸胍浓度的增大发生正触变性-复合触变性-负触变性的转变; 随盐酸胍作用时间的延长(6~48 h), 盐酸胍浓度为1.0 mol/L的胶原分散体系的触变性类型发生复合触变性-负触变性的转变. 本文的研究结果有助于加深对触变性产生机理的认识.     

Stability of proteins with multi-state unfolding behavior

A new model used to calculate the free energy change of protein unfolding is presented.In this model,proteins are considered to be composed of structural elements.The unfolding of a structural element obeys a two-state mechanism and the free energy change of the element can be obtained by a linear extrapolation method.If a protein consists of the same structural elements,its unfolding will displays a two-state process,and only the average structural element free energy change < △G 0 element(H2O)> can be measured.If protein consists of completely different structural elements,its unfolding will show a multi-state behavior.When a protein consists of n structural elements its unfolding will shows a(n+1)-state behavior.A least-squares fitting can be used to analyze the contribution of each structural element to the protein and the free energy change of each structural element can be obtained by using linear extrapolation to zero denaturant concentration,not to the start of each transition.The measured △G0 protein(H2 O) is the sum of the free energy change for each structural element.Using this new model,we can not only analyze the stability of various proteins with similar structure and similar molecular weight,which undergo multi-state unfolding processes,but also compare the stability of proteins with different structures and molecular weights using the average structural element free energy change < △G0 element(H2O)>.Although this method cannot completely provide the exact free energy of proteins,it is better than current methods.     

Thermodynamic analysis of lysozyme adsorbed to silica

The structural stability of hen egg white lysozyme in solution and adsorbed to small colloidal silica particles at various surface concentrations was investigated using hydrogen-deuterium (H/D) exchange in combination with mass spectrometry (HDX-MS) and differential scanning calorimetry (DSC). The combination of HDX-MS and DSC allows a full thermodynamic analysis of the lysozyme structure as both the enthalpy and the Gibbs free energy can be derived from the various measurements. Moreover, both HDX-MS and DSC provide information on the relative structural heterogeneity of lysozyme in the adsorbed state compared to that in solution. Results demonstrated that at high surface coverage, the structural stability of lysozyme was only marginally affected by adsorption to silica particles whereas the unfolding enthalpy decreased by more than 10%, meaning that the entropy of lysozyme increased with a similar value upon adsorption. Furthermore, the structural heterogeneity increased considerably. At lower surface concentrations, the structural heterogeneity increased further whereas the enthalpy of unfolding decreased. Further analyses of the HDX-MS experiments clearly indicated that folding/unfolding of lysozyme occurs through a two-domain process. These two domains had a similar amount of structural elements and a difference in stabilization energy of 8 kJ/mol, regardless if lysozyme was in solution or adsorbed to silica.     

虎纹捕鸟蛛毒素-Ⅴ去折叠过程的色谱分析

 将纯化的天然虎纹捕鸟蛛毒素-Ⅴ经盐酸胍变性30 min后,在pH 3.0、反应温度为37 ℃的条件下与三羧甲基磷酸(TCEP)反应12 min,用反相高效液相色谱分离得到其全部去折叠中间体,通过基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行鉴定,并利用烷基化反应对这些去折叠中间体予以进一步确证。 根据其保留时间,分析虎纹毒素-Ⅴ各去折叠中间体的色谱行为,初步探讨了多肽或蛋白质构象异构体反相色谱行为的多样性。     

荧光光谱法研究牛血清蛋白在盐酸胍体系中的变性

应用荧光光谱技术,对盐酸胍与牛血清蛋白在30℃水溶液中的结合作用及造成牛血清蛋白变性的过程进行了研究,考察了盐酸胍诱导牛血清蛋白变性时荧光强度和峰位的变化规律,并计算出伸展分数fu,变性平衡常数Ku,伸展吉布斯自由能△Gu,衡量蛋白质对变性剂稳定性的参量△GH2o,衡量蛋白质变性协同性的参量m和变性中点C1/2.研究结...     

Structural Stability and Unfolding Properties of Cutinases from Thermobifida fusca

A comparative analysis of the structural and functional aspects along with equilibrium unfolding of two homologous cutinases, Cut1 and Cut2, from Thermobifida fusca was carried out. The CD and fluorescence profile at different pH in the range of 6 to 9 showed no structural variations for both cutinases, indicating their stability to a wide range of pH. Tryptophan quenching studies suggested that all the four Trp residues in the protein are in inaccessible hydrophobic pockets. Further, near-UV CD analysis of tertiary structure revealed a dissimilar distribution of aromatic amino acid on the surface of these two enzymes. Denaturation profiles obtained in aqueous solutions of the guanidine hydrochloride revealed different tolerance levels for unfolding of the two cutinases, with Cut2 showing higher resistivity to unfolding in comparison to Cut1. Both cutinases retained all the structural parameters even in the presence of 8 M urea, indicating the protein to be highly resistant to urea-induced unfolding. Structural study by homology modeling revealed a high resemblance of secondary structure between the two cutinases; however, their tertiary structure, hydrophobicity, and surface electrostatic properties were very different, which contributed to the difference in the structural stability of these two cutinases.     

Simulation of the mechanical unfolding of ubiquitin: probing different unfolding reaction coordinates by changing the pulling geometry

Motivated by the recent experimental atomic force microscopy (AFM) measurements of the mechanical unfolding of proteins pulled in different directions [D. J. Brockwell et al., Nat. Struct. Biol. 10, 731 (2003); M. Carrion-Vazquez et al., ibid 10, 738 (2003)] we have computed the unfolding free energy profiles for the ubiquitin domain when it is stretched between its (A) N and C termini, (B) Lys48 and C terminus, (C) Lys11 and C terminus, and (D) N terminus and Lys63. Our results for cases (A) and (B) are in good agreement with the experimental unfolding forces measured for the N-C and Lys48-C linked polyubiquitin, in particular, indicating a considerably lower unfolding force in the latter case. Mechanical unfolding in case (A) involves longitudinal shearing of the terminal parallel strands while in case (C) the same strands are "unzipped" by the force. The computed unfolding forces in case (C) are found to be very low, less than 50 pN for pulling rates typical of AFM experiments. The unfolding free energy barrier found in case (C) is approximately 13 kcal/mol, which corresponds to a zero-force unfolding rate constant that is comparable to the rate of chemical unfolding extrapolated to zero denaturant concentration. The unfolding barrier calculated in case (A) in the limit of zero force is much higher, suggesting that mechanical unfolding in this case follows a pathway that is different from that of thermal/chemical denaturation.     

Structural Stability of Azurin Encapsulated in Sol-Gel Glasses: A Fluorometric Study

In this study we investigated the structural features of azurin, a blue copper-containing enzyme, upon encapsulation in tetramethoxysilane derived sol-gel glasses. Fluorescence spectroscopy revealed that gelation of inorganic networks does not affect the protein tertiary structure and only after two months solvent phase loss altered protein stability. In case of organically modified sol-gel matrices, the protein stability was reduced after encapsulation into hosts modified by adding 3-Mercaptopropyl-trimethoxysilane, 3-Glycidyloxypropyl-trimethoxysilane and Trimethoxy octylsilane, while it was found to be enhanced in networks doped with 3-Trimethoxysilyl-propyl methacrylate and 3-Aminopropyl-trimethoxysilane. In order to better investigate the effects of silica glasses on azurin stability, unfolding experiments of the protein, in solution or entrapped, were also performed in the presence of both methanol and guanidinium hydrochloride (GdHCl). Our results suggest that the matrix protects azurin against the aggregation induced by alcohol, and that the free energy change value upon unfolding by GdHCl was lower than the value calculated for azurin in solution and was dependent on the surface chemistry of silica matrix.