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...     

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。     

光谱法研究变性剂诱导的猪胃蛋白酶和牛血清蛋白折叠中间态的分布和过渡

利用紫外光谱和荧光光谱分别测定了变性剂诱导的猪胃蛋白酶和牛血清蛋白的残余活性,并根据它们的两个特征展开参数定量描述了猪胃蛋白酶和牛血清蛋白天然态、折叠中间态和完全展开态随变性液中变性剂浓度的分布和过渡。结果表明,在盐酸胍诱导的猪胃蛋白酶"三态"去折叠过程中,当盐酸胍浓度约为1.5mol/L时,折叠中间态的浓度达到最大,约占溶液中总猪胃蛋白酶的12%;在脲诱导的牛血清蛋白"三态"去折叠过程中,当脲浓度约为2.0mol/L时,折叠中间态的浓度达到最大,约占溶液中总牛血清蛋白的41%;而在盐酸胍诱导的牛血清蛋白"四态"去折叠过程中,当盐酸胍浓度约为0.4mol/L、1.2mol/L时,第一和第二折叠中间态浓度分别达到最大,约占溶液中总牛血清蛋白的20%和70%。     

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.     

荧光相图法研究猪胰腺α-淀粉酶在脲和盐酸胍溶液中的去折叠过程

用荧光相图法分别研究了脲和盐酸胍诱导的猪胰腺α-淀粉酶的去折叠过程。实验结果表明,当脲作为变性剂时,无论变性体系中有无还原剂2-巯基乙醇存在,猪胰腺α-淀粉酶的去折叠过程均只出现一个部分折叠中间体,符合“三态模型”;当盐酸胍作为变性剂时,若变性体系中存在还原剂2-巯基乙醇,猪胰腺α-淀粉酶的去折叠过程符合“三态模型”,而若变性体系中不存在还原剂2-巯基乙醇,猪胰腺α-淀粉酶的去折叠过程会出现两个部分折叠中间态,此过程符合“四态模型”。     

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.     

体积排阻色谱法研究变性溶菌酶分子复性过程中集聚体的形成

在体积排阻色谱柱上研究了还原剂存在时脲和盐酸胍变性的3种溶菌酶溶液的复性和分离过程。当变性液中原始溶菌酶浓度大于10 g/L时,变性溶菌酶在体积排阻色谱柱上除了复性为与未变性溶菌酶出峰时间相同的复性态溶菌酶分子外,还形成了溶菌酶折叠中间体的二分子集聚体。这个结果得到了用稀释法复性时溶菌酶的蛋白电泳检测结果的支持。与稀释法复性相比较,用体积排阻色谱法复性时所形成的折叠中间体二分子集聚体的量要远远低于用稀释法所形成的集聚体的量。     

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。     

蛋白质多态解折叠行为与稳定性Ⅱ:酶的多态解折叠行为

基于结构基元模型,进一步假设,由n个结构基元组成的蛋白酶,其活性中心由na(na?n)个结构基元组成,酶活性仅与组成活性中心的结构基元相关.由此,推导出适合于蛋白酶解折叠研究的变性曲线、解折叠结构基元平均自由能、物种分布等表达式.本文以盐酸胍诱导的卵清溶菌酶解折叠为例,通过荧光方法测定的溶菌酶解折叠曲线,得出卵清溶菌酶由2个结构基元组成,结构基元平均自由能?G0element(H2O)为48.47 kJ/mol.物种分布表明,酶活性随盐酸胍浓度的变化仅仅反映的是结构基元1(?-片结构域)的解折叠,而结构基元2(?-螺旋结构域)的解折叠反映在3.8~5.0 mol/L盐酸胍浓度范围内.结构基元模型既可描述蛋白酶多态解折叠的谱学行为,又可解释蛋白酶活性的两态性质.     

盐酸胍诱导的猪血红蛋白分子解离过程中各亚基的分布

以内源荧光光谱、荧光相图、高效凝胶排阻色谱和相对释氧曲线等方法,研究了盐酸胍诱导的猪血红蛋白分子解离过程中各亚基的分布状况.实验发现,当溶液中盐酸胍浓度小于3.5 mol/L时,猪血红蛋白分子仅仅以四聚体形式存在;而当溶液中盐酸胍浓度达到和超过3.5 mol/L时,部分猪血红蛋白分子开始从四聚体解离成二聚体和单体,并且...     

荧光相图法研究脲诱导牛血清白蛋白的去折叠过程

作为从分子水平上阐明生命奥秘的中心课题之一,蛋白质的折叠问题一直受到生物化学、生物物理学和结构生物学等领域研究工作者的高度关注。在蛋白质的变性过程中,它们往往达不到完全去折叠,而是会形成不同的部分折叠中间态[1-3],这些部分折叠中间态在蛋白质折叠过程中起着重要作     

Urea-induced Inactivation and Unfolding of Recombinant Phospholipid Hydroperoxide Glutathione Peroxidase from Oryza sativa

Phospholipid hydroperoxide glutathione peroxidase is an antioxidant enzyme that has the highest capability of reducing membrane-bound hydroperoxy lipids as compared to free organic and inorganic hydroperoxides amongst the glutathione peroxidases.In this study,urea-induced effects on the inactivation and unfolding of a recombinant phospholipid hydroperoxide glutathione peroxidase(PHGPx)from Oryza sativa were investigated by means of circular dichroism and fluorescence spectroscopy.With the increase of urea concentration,the residual activity of OsPHGPx decreases correspondingly.When the urea concentration is above 5.0 mol/L,there was no residual activity.In addition,the observed changes in intrinsic tryptophan fluorescence,the binding of the hydrophobic fluorescence probe ANS,and the far UV CD describe a common dependence on the concentration of urea suggesting that the conformational features of the native OsPHGPx are lost in a highly cooperative single transition.The unfolding process comprises of three zones:the native base-line zone between 0 and 2.5 mol/L urea,the transition zone between 2.5 and 5.5 mol/L urea,and the denatured base-line zone above 5.5 mol/L urea.The transition zone has a midpoint at about 4.0 mol/L urea.     

Spectral Studies on the Conformational Transitions of Bovine Insulin During Denaturant-induced Unfolding

Transitions among various molecule states and conformational changes of bovine insulin were investigated under different denaturing conditions by means of fluorescence phase diagrams,fluorescence quenching,1-anilinonaphthalene-8-sulfonate（ANS） binding assay and circular dichroism（CD） spectra.In both guanidine hydrochloride（GuHCl）-and urea-denatured procedures,the spatial structure of insulin molecules changed from ordered states to relative unordered ones with the increasing of denaturant concentration.The GuHCl-denatured process followed a four-state model,for there were two intermediates existed in 2.0 and 6.0 mol/L GuHC1,respectively.Intermediate I1 is more compact than the normal protein.And intermediate I2 has lost most of the secondary structures.When GuHCl concentration was above 6.0 mol/L,the fluorophores originally existed in the internal of insulin molecules would expose to the surface.However,the urea-denatured process followed a three-state model,only one intermediate existed in 2.5 mol/L urea.During the urea-denatured procedure,the fluorophores originally existed in theinternal of insulin molecules didn＇t expose to the surface.     

氧氟沙星与脲诱导牛血清白蛋白结合的机制研究

摘要 利用荧光光谱和紫外光谱研究了脲（Urea）对牛血清白蛋白（BSA）结构的影响以及氧氟沙星（Oflxacin）与脲诱导的BSA结合的情况。结果显示：Urea诱导BSA变性历经两步、三态过程,且伴随中间态的形成。随着Urea浓度的增大,BSA荧光强度降低并先蓝移（344 nm～336 nm）,后又红移至350 nm。Urea浓度在4.6～5.2 mol/L范围时,Oflx对BSA中间态有强的猝灭作用（KQ=10.46×104 L/mol, Urea 4.8 mol/L）和较大的结合常数（KA=3.8807×105 L/mol, Urea 4.8 mol/L）,但是结合位点数小（n=0.76, Urea 5.0 mol/L）,能量传递效率低（E=0.3002, Urea 4.8 mol/L）。同步荧光光谱显示：Urea诱导BSA去折叠时,Trp-212残基微环境并未发生改变,而Tyr的最大荧光发射峰蓝移,Oflx的加入诱导Trp-212的微环境更具疏水性。Oflx加速了Urea对BSA的失活作用。     

Electrochemically Monitoring the Acid and Acidic Urea‐Induced Unfolding of Hemoglobin and Its Electrocatalytic Ability

Acid and acidic urea‐induced unfolding of hemoglobin (Hb) was investigated using protein‐film‐based electrochemical method. The conformational transition of Hb was monitored through the change of direct electrochemical response of Hb. The heme groups in Hb detached from their native binding sites after pH<4.0. Spectral experiments fully supported the results. Furthermore, the catalytic ability of Hb was enhanced 15 times under the optimal unfolded conditions of pH 2.0 PBS containing 3.0 mol L^?1 urea. The method contributes to understand the relationship between function and conformation of Hb, and provides possibility of manipulating protein function by controlling its conformation.     

Identification of Folding Intermediates of Streblin,The Most Stable Serine Protease: Biophysical Analysis

Streblin, a serine proteinase from plant Streblus asper, has been used to investigate the conformational changes induced by pH, temperature, and chaotropes. The near/far UV circular dichroism activities under fluorescence emission spectroscopy and 8-aniline-1-naphthalene sulfonate (ANS) binding have been carried out to understand the unfolding of the protein in the presence of denaturants. Spectroscopic studies reveal that streblin belongs to the α+β class of proteins and exhibits stability towards chemical denaturants, guanidine hydrochloride (GuHCl). The pH-induced transition of this protein is noncooperative for transition phases between pH 0.5 and 2.5 (midpoint, 1.5) and pH 2.5 and 10.0 (midpoint, 6.5). At pH 1.0 or lower, the protein unfolds to form acid-unfolded state, and for pH 7.5 and above, protein turns into an alkaline denatured state characterized by the absence of ANS binding. At pH 2.0 (1 M GuHCl), streblin exists in a partially unfolded state with characteristics of a molten globule state. The protein is found to exhibit strong and predominant ANS binding. In total, six different intermediate states has been identified to show protein folding pathways.     

A designed well-folded monomeric four-helix bundle protein prepared by Fmoc solid-phase peptide synthesis and native chemical ligation

The design and total chemical synthesis of a monomeric native-like four-helix bundle protein is presented. The designed protein, GTD-Lig, consists of 90 amino acids and is based on the dimeric structure of the de novo designed helix-loop-helix GTD-43. GTD-Lig was prepared by the native chemical ligation strategy and the fragments (45 residues long) were synthesized by applying standard fluorenylmethoxycarbonyl (Fmoc) chemistry. The required peptide-thioester fragment was prepared by anchoring the free gamma-carboxy group of Fmoc-Glu-allyl to the solid phase. After chain elongation the allyl moiety was orthogonally removed and the resulting carboxy group was functionalized with a glycine-thioester followed by standard trifluoroacetic acid (TFA) cleavage to produce the unprotected peptide-thioester. The structure of the synthetic protein was examined by far- and near-UV circular dichroism (CD), sedimentation equilibrium ultracentrifugation, and NMR and fluorescence spectroscopy. The spectroscopic methods show a highly helical and native-like monomeric protein consistent with the design. Heat-induced unfolding was studied by tryptophan absorbance and far-UV CD. The thermal unfolding of GTD-Lig occurs in two steps; a cooperative transition from the native state to an intermediate state and thereafter by noncooperative melting to the unfolded state. The intermediate exhibits the properties of a molten globule such as a retained native secondary structure and a compact hydrophobic core. The thermodynamics of GuHCl-induced unfolding were evaluated by far-UV CD monitoring and the unfolding exhibited a cooperative transition that is well-fitted by a two-state mechanism from the native to the unfolded state. GTD-Lig clearly shows the characteristics of a native protein with a well-defined structure and typical unfolding transitions. The design and synthesis presented herein is of general applicability for the construction of large monomeric proteins.     

Effect of Native Disulfide Bonds on the Denaturation of Proteins in 6 mol/L Guanidinium Chloride——A Fourier Transformed Infrared Study

The unfolding of bovine serum albumin, lysozyme and ribonuclease A denatured in 6 mol/L GuHCl with their disulfide bridges intact and reduced have been compared by FTIR studies. The peak positions and heights in the deconvolved spectra of amide I bands of the above denatured proteins with native disulfide bonds show marked differences whereas those for the denatured proteins without disulfide linkages are closely similar. The above and other evidence suggest that denatured proteins with intact disulfides still have considerable ordered conformation even in 6 mol/L GuHCl.