新天花粉蛋白的盐酸胍去折叠过程

本文利用荧光光谱和园二色光谱研究了新天花粉蛋白的盐酸胍去折叠过程.结果显示:新天花粉蛋白的盐酸胍去折叠是一个只包含天然蛋白和变性终态的二态过程,与已经报道的天花粉蛋白的盐酸胍去折叠过程不同.     

基于光谱法研究盐酸胍诱导的人血红蛋白去折叠过程

借助于紫外-可见吸收光谱法、荧光光谱法以及停流-荧光光谱法研究了盐酸胍(GdmHcl)诱导人血红蛋白的去折叠过程。实验发现,盐酸胍诱导的血红蛋白去折叠有两个不同的过程,即随着GdmHcl浓度增加到1.0 mol·L-1左右时,血红蛋白亚基发生解聚,形成中间态;持续增加其浓度时,各亚基发生内部去折叠,最终导致血红素发生崩解。加入还原剂(β-巯基乙醇)对血红蛋白亚基解聚、血红素崩解有协同作用且直接引起亚基和全分子同步变构。血红蛋白去折叠过程从“三态模型”转变为“二态模型”。     

硫酸铜诱导烟草多酚氧化酶抗变性的研究

为了进一步探讨外源铜与多酚氧化酶的相互作用 ,通过酶活性测定 ,荧光光谱和圆二色光谱 (CD)对外源Cu2 + 诱导烟草多酚氧化酶抗变性进行了研究 .荧光光谱结果表明 ,没有外源铜存在的情况下 ,盐酸胍诱导的烟草多酚氧化酶变性是一个二态过程 ,烟草多酚氧化酶在 6mol/L盐酸胍中变性 5min ,酶活性降至 18.6 % ,变性30min便彻底失活 ;10 .0mmol/L外源铜存在时 ,盐酸胍诱导的烟草多酚氧化酶变性是一个三态过程 ,上述相同盐酸胍浓度下变性相同时间 ,酶的剩余活性分别为 6 0 .6 %和 2 4 .9% .CD谱数据结果表明 ,6mol/L盐酸胍中变性 30min ,烟草PPO二级结构中α 螺旋、反平行 β 折叠、β 转角 /平行 β 折叠、芳香残基和二硫键、无规卷曲 /γ 转角的百分含量分别为 1.1、3.8、3.3、7.5和 84 .3;在 10 .0mmol/LCu2 + 存在时 ,上述相同变性条件下 ,各种二级结构百分含量分别为 34.2、13.7、2 1.0、9.5和 2 1.6 .适量外源铜可明显提高烟草多酚氧化酶结构的稳定性 .     

荧光光谱法研究盐酸胍浓度不同时变性胰蛋白酶的构象变化

蛋白变性过程中间体的存在是蛋白变性及复性动力学研究中不可缺少的证据。以胰蛋白酶为模型蛋白 ,用荧光光谱法系统地研究了在不同浓度变性剂盐酸胍存在时胰蛋白酶构象的变化 ,并与活性数据进行了对比。发现胰蛋白酶荧光光谱发射波长随变性剂盐酸胍浓度增大而逐渐增大 ,并且当盐酸胍浓度达到 2mol·L- 1 时胰蛋白酶的最大发射波长达到最大值 ,其后随盐酸胍浓度的增大最大发射波长反而逐渐减小 ,当盐酸胍浓度大于 3mol·L- 1 呈现不变的趋势。也就是说 ,在低浓度变性剂环境下 ,胰蛋白酶存在着一个与天然态和完全变性态的分子构象都不同的中间体状态 ,这个中间体状态的荧光发射波长最大 ,荧光发射强度也最大 ,而以此状态为复性起点 ,最终得到的复性产率也最低。对此原因从分子结构的基础上进行了探讨。     

FTIR光谱法研究天花粉蛋白的热去折叠过程

本文利用ＦＴＩＲ光谱技术和计算机辅助解析技术（二阶导数、去卷积和曲线拟合）研究了天花粉蛋白的热诱导去折叠过程。结果表明：在２５～８５℃温度范围内,天花粉蛋白的热去折叠是一个不可逆的分子间聚集的过程;二级结构随温度的变化暗示了折叠中间体的存在。     

热诱导β晶状体蛋白去折叠及寡聚体解聚过程的脉冲升温时间分辨红外光谱

通过变温傅里叶变换红外光谱和脉冲升温时间分辨红外光谱,系统地研究了β晶状体蛋白不同二级结构的热稳定性,以及热诱导β晶状体蛋白去折叠的动力学过程．结果表明,β晶状体蛋白N端的β反平行折叠热稳定性最低(转变的中点温度为36.0±2.1 oC),该结构的破坏导致了一种富含无规卷曲结构的β晶状体蛋白中间体形成, 认为该中间体可能由β晶状体寡聚体解聚后形成的单体,单体形成的中点温度为40.4±7 πC．β晶状体蛋白全局去折叠引发蛋白变性聚集的转变的中点温度为72.4±0.2 oC．脉冲升温时间分辨红外光谱进一步揭示出β晶状体蛋白的热诱导去折叠开始于N端的β反平行折叠结构,该结构去折叠所需时间约为50 ns     

肌红蛋白力致去折叠的全原子分析

位于脊椎动物骨骼肌中的肌红蛋白在生命过程中扮演着重要角色，其折叠过程依赖于血红素的绑定.本文着重于肌红蛋白在力的作用下去折叠过程的全原子统计分析.结果表明血红素不仅具有生物学上的功能，而且决定其去折叠的动力学过程.在血红素不存在的情况下，肌红蛋白的力致去折叠过程存在一个中间态，其构象不同于化学变性剂所导致的去折叠中间态，发现新的中间态.结论与相关实验结果相符，揭示了肌红蛋白力致去折叠的一般机制.     

肌红蛋白力致去折叠的全原子分析（英文）

位于脊椎动物骨骼肌中的肌红蛋白在生命过程中扮演着重要角色,其折叠过程依赖于血红素的绑定.本文着重于肌红蛋白在力的作用下去折叠过程的全原子统计分析.结果表明血红素不仅具有生物学上的功能,而且决定其去折叠的动力学过程.在血红素不存在的情况下,肌红蛋白的力致去折叠过程存在一个中间态,其构象不同于化学变性剂所导致的去折叠中间态,发现新的中间态.结论与相关实验结果相符,揭示了肌红蛋白力致去折叠的一般机制.     

荧光光谱法研究氨基改性介孔泡沫对DhaA的稳定化机理

采用F-4600荧光光谱仪,对尿素和二甲基亚砜两种变性剂中烷基卤脱卤酶DhaA在氨基改性介孔泡沫固定化前后的荧光光谱特征进行测定。运用荧光相图分析DhaA在两种变性剂中的去折叠过程,并结合活性残留率进行了变性过程热力学参数计算,比较固定化前后DhaA去折叠过程和热力学参数的区别。实验结果表明,DhaA催化活性随变性剂浓度增加而降低。相同变性剂浓度下,固定化DhaA能够比游离态DhaA保持更高的催化活性,在变性剂到达临界浓度之前（尿素浓度5.5 mol·L-1,DMSO浓度7 mol·L-1）,氨基改性介孔泡沫的稳定化作用显著。DhaA在尿素诱导下的变性过程符合“二态模型”,而在DMSO诱导下符合“三态模型”,DhaA中间态出现在浓度为5.6 mol·L-1。氨基改性介孔泡沫固定化不改变DhaA变性过程,但能够提高DhaA的去折叠热力学参数。在尿素诱导下,计算得到的DhaA初始吉布斯自由能变ΔG(H₂O)为8.51 kcal·mol-1,固定化后ΔG(H₂O)提高为9.55 kcal·mol-1;但由于尿素分子容易通过静电作用进入氨基介孔泡沫孔道,固定化后DhaA的溶液可及面积m由3.69 kcal·(mol·mol·L-1)-1增大到4.00 kcal·(mol·mol·L-1)-1,孔道内的氨基、羟基能够通过氢键作用增强DhaA的刚性,从而有效的降低了尿素可及面积增加带来的影响,提高了DhaA的尿素耐受性。在DMSO诱导下,计算发现游离态与固定化DhaA在折叠态向中间态转变过程中的ΔG(H₂O)均为12.12 kcal·mol-1,由于孔道内的氨基、羟基能够有效阻碍非极性DMSO分子的进入,造成m从3.39 kcal·(mol·mol·L-1)-1降低为2.30 kcal·(mol·mol·L-1)-1;当DhaA从中间态向去折叠态转化时,DhaA内部疏水基团暴露导致m增加,由于孔道内极性微环境作用,固定化DhaA的m值（4.40 kcal·(mol·mol·L-1)-1）仍然低于游离态DhaA（4.94 kcal·(mol·mol·L-1)-1）。荧光光谱法研究固定化对DhaA去折叠过程及热力学参数的影响是深入研究DhaA稳定性的有效手段,能够为其他生物酶的稳定化机理研究提供方法指导。     

不同处理条件对乳铁蛋白构象的影响研究

运用荧光、圆二色谱及紫外光谱手段,研究了六种不同物理或化学条件处理后,牛乳铁蛋白(Bovine lactoferrin,bLF)三级、二级结构及二硫键的变化。荧光结果显示:6mol.L-1盐酸胍、8mol.L-1尿素和50mmol.L-1二硫苏糖醇三种处理后bLF溶液的最大发射波长从333nm红移至354nm,疏水基团大量暴露,三级结构发生明显变化;100℃加热5min、超声(450W,5s,6个脉冲)、1%巯基乙醇处理后,bLF溶液荧光强度明显减弱,最大发射波长几乎无变化。圆二色谱结果表明:经盐酸胍处理,bLF中α-螺旋结构消失,其余五种处理,二级结构的变化较小。紫外光谱数据表明:二硫苏糖醇对bLF二硫键破坏最严重,超过总二硫键含量的55%,超声次之,盐酸胍、巯基乙醇和加热破坏较少。结果对进一步明确乳铁蛋白的构效关系提供了一定的依据。     

Conformational and Functional Transitions in Class II α-mannosidase from <Emphasis Type="Italic">Aspergillus fischeri</Emphasis>

The conformational transitions in an oligomeric and high molecular weight class II α-mannosidase from Aspergillus fischeri were examined using fluorescence and CD spectroscopy under chemical, thermal and acid denaturing conditions. The enzyme lost the activity first and then the overall folded conformation and secondary structure. The midpoint values of GdnHCl mediated changes measured by inactivation; fluorescence and negative ellipticity were 0.48 M, 1.5 M and 1.9 M, respectively. The protein almost completely unfolded in 4.0 M GdnHCl but not at 90 °C. The inactivation and unfolding were irreversible. At pH 2.0, the protein exhibited molten-globule like intermediate with rearranged secondary and tertiary structures and exposed hydrophobic amino acids on the surface. This species showed increased accessibility of Trp to the quenchers and got denatured with GdnHCl in a different manner. The insoluble aggregates of a thermally denatured protein could be detected only in the presence of 0.25–0.75 M GdnHCl.     

醇引起酵母醇脱氢酶构象变化的光谱研究

应用紫外、荧光、ＣＤ谱研究了酵母醇脱氢酶经乙醇、正丙醇和乙二醇作用后的构象变化。结果表明２２０ｎｍ、２８０ｎｍ处的紫外吸收，３３６ｎｍ的相对荧光强度随醇浓度增大而加强。ＣＤ谱显示乙醇、乙二醇引起酶分子在２０８ｎｍ、２２０ｎｍ处的双负峰逐渐加强，正丙醇使２２０ｎｍ处负峰加强，２０８ｎｍ处负峰减弱并红移，直至完全消失。上述数据表明醇浓度增加导致酶分子结构逐步松散，同时伴随着活力的丧失。     

The Detection of Unfolding Intermediates of Soybean Lipoxygenase-1 during Urea Denaturation by Fluorescence Spectroscopy

The unfolding of soybean lipoxygenase-1 during urea denaturation has been followed by activity assays and fluorescence measurement. The presence of stable intermediates during unfolding for both ferrous and ferric forms of lipoxygenase-1 were observed. In the presence of 6.0 M urea, the unfolding of soybean lipoxygenase-1, as monitored by fluorescence intensity, is a triphasic process, while the inactivation of the enzyme shows a single-phase kinetics. The rate constant of inactivation is consistent with that of the fast conformational change of the enzyme. Based on these, a minimal scheme containing two intermediates was proposed to interpret the unfolding of lipoxygenase-1 induced by urea.     

Interaction between silver nanoparticle and bovine hemoglobin at different temperatures

The binding of silver nanoparticles to bovine hemoglobin (BHb) was studied by fluorescence, UV–Visible, and circular dichroism (CD) spectroscopic techniques at different temperatures of 20, 37, and 42 °C. The absorption spectrum of soret band, in the presence of silver nanoparticle, showed a significant spectral change, which indicated the heme groups of BHb were directly attacked and degraded by silver nanoparticle. The fluorescence data explained that the nanoparticle binding to BHb occurred at a single binding site, which demonstrated a dynamic quenching procedure. Nanoparticles could reduce the fluorescence of tryptophanyl residues of BHb to a lesser extent. Circular dichroism studies demonstrated a conformational change of BHb in the presence of silver nanoparticles. The helicity of BHb was reduced by increasing silver nanoparticle concentration at different temperatures. Thermodynamic analysis of the protein interaction by silver nanoparticles suggested that the binding process is only entropy driven.     

Mössbauer spectroscopic evidence on the heme binding to the proximal histidine in unfolded carbonmonoxy myoglobin by guanidine hydrochloride

The unfolded heme structure in myoglobin is controversial because of no chance of direct X-ray structure analyses. The unfolding of carbonmonoxy myoglobin (MbCO) by guanidine hydrochloride (GdnHCl) was studied by the Mössbauer spectroscopy. The spectra show the presence of a sort of spectrum in the unfolded MbCO, independent on the concentration of GdnHCl from 1 to 6 M and the increase of the fraction of unfolded MbCO, depending on the GdnHCl concentration. The isomer shift of the iron of heme in the unfolded MbCO was identified to be different from that of the native MbCO as the globin structure in Mb collapses under the unfolded conditions. This result and the existing related Mössbauer data proved that the heme in the unfolded MbCO may remain coordinated to the proximal histidine.     

Effects of ultrasound and ultrasound assisted alkaline pretreatments on the enzymolysis and structural characteristics of rice protein

The objectives of this study were to investigate the effects of multi-frequency energy-gathered ultrasound (MFEGU) and MFEGU assisted alkaline pretreatments on the enzymolysis and the mechanism of two pretreatments accelerating the rice protein (RP) proteolysis process. The results showed that MFEGU and MFEGU assisted alkaline pretreatments improved significantly (P < 0.05) the degree of hydrolysis (DH) and the protein elution amount of RP. Furthermore under the same DH conditions, ultrasound and ultrasound assisted alkaline pretreatments were more save the enzymolysis time than the unpretreatment. The changes in UV–vis spectra, fluorescence emission spectra indicated unfolding and destruction of RP by MFEGU and MFEGU assisted alkaline pretreatments. The circular dichroism analysis showed that both pretreatments decreased α-helix but increased β-sheet and random coil of RP. Amino acid composition revealed that MFEGU and MFEGU assisted alkaline pretreatments could increase the protein elution amount and the ratio of hydrophobic amino acids. Atomic force microscopy (AFM) indicated that both pretreatments destroyed the microstructures and reduced the particle size of RP. Therefore, MFEGU and MFEGU assisted alkaline pretreatments are beneficial to improving the degree of hydrolysis due to its sonochemistry effect on the molecular conformation as well as on the microstructure of protein.     

Complexation of insecticide chlorantraniliprole with human serum albumin: Biophysical aspects

Chlorantraniliprole is a novel insecticide belonging to the diamide class of selective ryanodine receptor agonists. A biophysical study on the binding interaction of a novel diamide insecticide, chlorantraniliprole, with staple in vivo transporter, human serum albumin (HSA) has been investigated utilizing a combination of steady-state and time-resolved fluorescence, circular dichroism (CD), and molecular modeling methods. The interaction of chlorantraniliprole with HSA gives rise to fluorescence quenching through static mechanism, this corroborates the fluorescence lifetime outcomes that the ground state complex formation and the predominant forces in the HSA-chlorantraniliprole conjugate are van der Waals forces and hydrogen bonds, as derived from thermodynamic analysis. The definite binding site of chlorantraniliprole in HSA has been identified from the denaturation of protein, competitive ligand binding, and molecular modeling, subdomain IIIA (Sudlow's site II) was designated to possess high-affinity binding site for chlorantraniliprole. Moreover, using synchronous fluorescence, CD, and three-dimensional fluorescence we testified some degree of HSA structure unfolding upon chlorantraniliprole binding.     

利用内禀荧光探针研究蛋白相互作用中硫氧还蛋白的氧化还原态

细胞内蛋白质的氧化还原状态直接影响细胞的增殖、分化及凋亡,而氧化还原状态的改变对调控细胞的生存或死亡尤为重要。硫氧还蛋白(Thioredoxin, TRX)是一种广泛存在于生物体内的氧化还原调节蛋白,其在细胞内氧化还原状态的变化是发挥其氧化还原调控作用的重要过程。以TRX为对象并以其中的色氨酸残基(Trp)作为内禀荧光探针,利用蛋白质定点突变、SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)、荧光光谱和圆二色谱等技术和方法,研究TRX与谷胱甘肽过氧化物酶(glutathione peroxidase, GPX3)相互作用过程中氧化还原态的变化。通过观测TRX以及突变体中色氨酸荧光光谱的变化,研究蛋白相互作用的电子转移模式以及TRX氧化态-还原态之间的相互转化。结果表明氧化态的TRX与还原态的GPX3之间存在相互作用并发生电子交换,解释了二者之间电子传递模式为GPX3将电子传递给TRX,为揭示TRX在细胞信号传递过程中的物理化学机制提供了实验依据。