脱血红素细胞色素C自发折叠的进一步验证

用胰蛋白酶水解结构聚丙烯酰胺凝胶电泳以及纳秒内源荧光衰减谱分析对鸡心脱血红素细胞色素Ｃ在透析复性过程的自发折叠现象作进一步确定，结果显示随着透析复性时间的增加，鸡心脱血红素细胞色素Ｃ对胰蛋白酶的水解敏感性逐渐下降，内源荧光寿命值增大，与此对应的马心脱血红素细胞色素Ｃ没有发生变化。     

脱血红素细胞色素c自发折叠的进一步验证

用胰蛋白酶水解结合聚丙烯酰胺凝胶电泳以及纳秒内源荧光衰减谱分析对鸡心脱血红素细胞色素ｃ在透析复性过程的自发折叠现象作进一步确定。结果显示随着透析复性时间的增加,鸡心脱血红素细胞色素ｃ对胰蛋白酶的水解敏感性逐渐下降,内源荧光寿命值增大,与此对应的马心脱血红素细胞色素ｃ没有发生变化。     

不同解折叠状态鸡心脱血红素细胞色素c分子的表面性质

脱血红素细胞色素ｃ的构象与其跨膜转运能力是密切相关的。鸡心脱血红素细胞色素ｃ具有较强的自发折叠的能力,在一定条件下可以得到不同解折叠状态的鸡心脱血红素细胞色素ｃ。本文利用疏水层析、与疏水探针１,８—ＡＮＳ的结合以及色氨酸与结合的ＡＮＳ之间的荧光共振能量转移,比较了不同解折叠状态的鸡心脱血红素细胞色素ｃ分子的表面性质。结果表明,部分折叠的鸡心脱血红素细胞色素ｃ分子获得了一种高度动态的结构,形成了动态的疏水核心;同时,它也具有较强的凝集倾向。这些性质与鸡心脱血红素细胞色素ｃ分子较强的自发折叠能力是一致的,为进一步分析鸡心脱血红素细胞色素ｃ分子的构象及其在跨膜转运过程中与脂的相互作用奠定了基础。     

脱血红素细胞色素c插入磷脂单分子层能力的研究

介绍了一种改进的、用于研究膜脂-蛋白相互作用的气-液界面单分子层实验模型及实验装置,并在该实验装置上研究了来自马心和金枪鱼心的线粒体前体蛋白脱血红素细胞色素c与大豆磷脂单分子层的相互作用,实验结果表明这两种前体蛋白对大豆磷脂单分子层都具有较强的亲和性和插膜能力,其临界插膜压力分别为43mN/m、45mN/m.     

脱血红素细胞色素c与膜结合及插膜时的构象研究

应用特殊的单分子层样品制备技术,分别制备了与中性、酸性磷脂膜结合的和完全插膜的鸡心脱血红素细胞色素c样品,并运用圆二色谱(CD)、表面衰减全反射Fourier变换红外光谱(ATR-FTIR)对膜上蛋白的构象进行了鉴定.研究结果表明,蛋白在与膜结合及插入阶段的构象是不同的,膜界面性质的不同也会对蛋白的构象产生不同的诱导,在酸性磷脂DSPG膜表面,该蛋白是以α螺旋和β折叠混合的构象形式结合;而在中性磷脂DSPC膜表面是以β折叠为主的构象形式结合.插入 DOPG单分子层内时则是 α螺旋为主的构象形式.     

C17S和H18D突变对鸡脱辅基细胞色素c跨线粒体膜输入的影响

体外转录鸡脱辅基细胞色素c(apocytochromec,简称apocyt.c)mRNA ,以之翻译apocyt.c并以3 5 S 甲硫氨酸标记 ,在纯化的鸡心线粒体上对它的跨膜转运与其经血红素加合酶催化转化为细胞色素c的关系进行了研究 .结果表明 ,即使在不利于形成细胞色素c的生化条件下鸡apocyt.c也能有效地输入线粒体 .为进一步证实apocyt.c的跨膜转运过程独立于转化为细胞色素c ,对apocyt.c的血红素结合位点进行基因的定点突变 ( 1 7位 :Cys→Ser;1 8位 :His→Asp) ,然后研究了 2个突变体apocyt.c的跨膜转运 .结果C1 7S和H1 8D都仍能有效地输入线粒体 ,但发现转运初速率已显著变慢     

脱血红素细胞色素c对DMPG膜的插入研究

气－液界面单分子层实验发现鸡心脱血红素细胞色素ｃ对ＤＭＰＧ单分子层表现出很强的插入能力,这种插入能力受亚相溶液离子强度的调节。通过傅里叶变换红外光谱对鸡心脱血红素细胞色素ｃ与ＤＭＰＧ脂质体的作用进行了进一步研究,对磷脂不同区域的红外光谱分析表明鸡心脱血红素细胞色素ｃ不仅与ＤＭＰＧ头部存在静电相互作用而且与其疏水部分也存在相互作用。     

不同折叠状态的Apocyt．C插膜后构象的研究

比较了不同折叠状态的鸡心脱血红素细胞色素ｃ（Ａｐｏｃｙｔ．ｃ）对大豆磷脂单分子层的插入能力和对酸性、中性磷脂单分子层的插入能力。不同折叠状态的Ａｐｏｃｙｔ．ｃ与大豆磷脂脂质体作用后的内源荧光发射谱提示它们各自在膜上的构象状态不同。与纯磷脂脂质体作用后的圆二色谱发现,溶液中折叠状态不同的鸡心Ａｐｏｃｙｔ．ｃ与膜作用后的构象也不同,溶液中处于无规心Ａｐｏｃｙｔ．ｃ与ＤＭＰＧ脂质体作用后的构象也呈α螺旋,但螺旋含量明显低于前者。折叠状态的鸡心Ａｐｏｃｙｔ．ｃ与ＤＭＰＣ作用后,其构象几乎没有变化。     

去血红素细胞色素c的制备及其结构研究

在酸性条件下用硫酸银断裂马心细胞色素ｃ（以下简称ｃｙｔ．ｃ）的肽链与血红素相连的硫醚键,通过酸性丙酮抽提,巯基乙醇处理及超速离心等步骤纯化得去血红素的ｃｙｔ．ｃ（以下简称Ａｐｏ－ｃｙｔ．ｃ．）．Ａｐｏ－ｃｙｔ．ｃ与天然ｃｙｔ．ｃ相比,其酸性电泳迁移率明显降低,紫外－可见光谱在１９０￣２２０ｎｍ处吸收上升,荧光光谱的最大发射峰波长产生红移,同时ＣＤ谱中ａ螺旋的特征峰完全消失,这说明在ｃｙｔ．ｃ去血红     

脱辅基细胞色素c定向于线粒体的转运特异性研究

用分离纯化的完整线粒体和部分细胞器组分,初步研究了脱辅基细胞色素c在细胞内转运的特异性。完整线粒体用差速离心和密度梯度离心的方法,从幼龄鸡心肌组织中获得,对胞内几种细胞器标志酶比活力的测量表明,纯化的线粒体单胺氧化酶活力提高25.6倍,腺苷酸激酶活力提高3.59倍,细胞色素c氧化酶活力提高5.48倍,外膜完整性达90%以上,呼吸控制率大于20。以上数据表明该纯化的线粒体受胞内其它囊泡成分污染少,外膜完整并具有较高的氧化磷酸化偶联效率;在纯化线粒体的同时,得到另两种细胞器组分-内质网和溶酶体囊泡。体外转录翻译的apo.c与上述几个组分的结合实验表明,完整线粒体与apo.c的结合能力明显高于其它组分。     

Role of Protein Stabilizers on the Conformation of the Unfolded State of Cytochrome c and Its Early Folding Kinetics: INVESTIGATION AT SINGLE MOLECULAR RESOLUTION*

An insight into the conformation and dynamics of unfolded and early intermediate states of a protein is essential to understand the mechanism of its aggregation and to design potent inhibitor molecules. Fluorescence correlation spectroscopy has been used to study the effects of several model protein stabilizers on the conformation of the unfolded state and early folding dynamics of tetramethyl rhodamine-labeled cytochrome c from Saccharomyces cerevisiae at single molecular resolution. Special attention has been given to arginine, which is a widely used stabilizer for improving refolding yield of different proteins. The value of the hydrodynamic radius (r_H) obtained by analyzing the intensity fluctuations of the diffusing molecules has been found to increase in a two-state manner as the protein is unfolded by urea. The results further show that the presence of arginine and other protein stabilizers favors a relatively structured conformation of the unfolded states (r_H of 29 Å) over an extended one (r_H of 40 Å), which forms in their absence. Also, the time constant of a kinetic component (τ_R) of about 30 μs has been observed by analyzing the correlation functions, which represents formation of a collapsed state. This time constant varies with urea concentration representing an inverted Chevron plot that shows a roll-over and behavior in the absence of arginine. To the best of our knowledge, this is one of the first applications of fluorescence correlation spectroscopy to study direct folding kinetics of a protein.     

四氯合铂酸钾(K_2PtCl_4)与细胞色素c(Cyt·c)修饰体系的进一步研究

从K_2PtCl_4与Cyt·c反应产物中分离得四个组分,SDS-PAGE及元素分析测定结果表明,它们分别对应于Cyt·c寡聚体、三聚体、二聚体及单体。这揭示了K_2PtCl_4对Cyt·c存在多位点修饰,南非如Kostic报道的Met-65为其唯一修饰位点。通过K_2PtCl_4与羧甲基化Cyt·c修饰产物的分析研究,证实了另一标记位点His-33的存在。对Cyt·c二聚体衍生物的还原电位、紫外一可見及红外光谱研究表明,Cyt·c血红素周围的电子环境仍保持完整,但其二级结构有较大的扰动。     

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX₉C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX₉C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.     

蛋白质折叠速度与其拓扑结构关系的研究

以6种不同的方式来定义蛋白质内存在的接触,进而运用分子动力学模拟等不同方法,对10个小蛋白进行分析,研究了不同的接触定义及不同的拓扑参数计算方法下,蛋白质的折叠速度与其拓扑参数的关系.结果表明,用含主链重原子的方式定义接触,所计算的拓扑参数与蛋白质折叠速度的相关性较好;用含侧链原子的方式定义接触,得到的拓扑参数与β型蛋白质的折叠速度的相关性较好.对不同的蛋白质,其拓扑结构与相应折叠速度间的相关程度不同。     

The Effect of Electrostatics on the Marginal Cooperativity of an Ultrafast Folding Protein

Proteins fold up by coordinating the different segments of their polypeptide chain through a network of weak cooperative interactions. Such cooperativity results in unfolding curves that are typically sigmoidal. However, we still do not know what factors modulate folding cooperativity or the minimal amount that ensures folding into specific three-dimensional structures. Here, we address these issues on BBL, a small helical protein that folds in microseconds via a marginally cooperative downhill process (Li, P., Oliva, F. Y., Naganathan, A. N., and Muñoz, V. (2009) Proc. Natl. Acad. Sci. USA. 106, 103–108). Particularly, we explore the effects of salt-induced screening of the electrostatic interactions in BBL at neutral pH and in acid-denatured BBL. Our results show that electrostatic screening stabilizes the native state of the neutral and protonated forms, inducing complete refolding of acid-denatured BBL. Furthermore, without net electrostatic interactions, the unfolding process becomes much less cooperative, as judged by the broadness of the equilibrium unfolding curve and the relaxation rate. Our experiments show that the marginally cooperative unfolding of BBL can still be made twice as broad while the protein retains its ability to fold into the native three-dimensional structure in microseconds. This result demonstrates experimentally that efficient folding does not require cooperativity, confirming predictions from theory and computer simulations and challenging the conventional biochemical paradigm. Furthermore, we conclude that electrostatic interactions are an important factor in determining folding cooperativity. Thus, electrostatic modulation by pH-salt and/or mutagenesis of charged residues emerges as an attractive tool for tuning folding cooperativity.     

Quinones in chlorosomes of green sulfur bacteria and their role in the redox-dependent fluorescence studied in chlorosome-like bacteriochlorophyll c aggregates

The light-harvesting chlorosome antennae of anaerobic, photosynthetic green sulfur bacteria exhibit a highly redox-dependent fluorescence such that the fluorescence intensity decreases under oxidizing conditions. We found that chlorosomes from Chlorobium tepidum contain three isoprenoid quinone species (chlorobiumquinone, menaquinone-7, and an unidentified quinone that probably is a chlorobiumquinone derivative) at a total concentration of approximately 0.1 mol per mol bacteriochlorophyll c. Most of the cellular chlorobiumquinone was found in the chlorosomes and constituted about 70% of the total chlorosome quinone pool. When the quinones were added to artificial, chlorosome-like bacteriochlorophyll c aggregates in an aqueous solution, a high redox dependency of the fluorescence was observed. Chlorobiumquinones were most effective in this respect. A lesser redox dependency of the fluorescence was still observed in the absence of quinones, probably due to another unidentified redox-active component. These results suggest that quinones play a significant, but not exclusive role in controlling the fluorescence and in inhibiting energy transfer in chlorosomes under oxic conditions. Chlorosomes from Chloroflexus aurantiacus contained menaquinone in an amount similar to that of total quinone in Chlorobium tepdium chlorosomes, but did not contain chlorobiumquinones. This may explain the much lower redox-dependent fluorescence observed in Chloroflexus chlorosomes. Received: 4 November 1996 / Accepted: 18 February 1997     

缢蛏碱性磷酸酶胍溶液中分子折叠与活力变化研究

报道了缢蛏碱性磷酸酶（简称ＡＬＰ）经不同浓度盐酸胍处理时酶的分子构象所发生的变化以及酶变化和失活的动力学过程。在胍中酶荧光发射峰强度下降,紫外差光谱在２４６ｎｍ和２８５ｎｍ处出现２个负峰,ＣＤ谱中酶的α螺旋度下降,且随浓度增大,变化程度也加大。动力学研究表明,酶在０．５ｍｏｌ／Ｌ、１．０ｍｏｌ／Ｌ、２．０ｍｏｌ／Ｌ３．０ｍｏｌ／Ｌ、４．０ｍｏｌ／Ｌ盐酸胍中的变性速度常数分别为３．２１×１０~（－４）ｓ~（－１）、６．３８×１０~（－４）ｓ~（－１）、２．１７×１０~（－３）ｓ~（－１）、２．３３×１０~（－３）ｓ~９－１）、５．１７×１０~（－３）ｓ~（－１）;而酶在相应盐酸胍中的失活速度常数分别为２．３３×１０~（－４）ｓ~（－１）、３．５７×１０~（－４）ｓ~（－１）、５．８６×１０~（－４）ｓ~（－１）、１．１４×１０~（－３）ｓ~（－１）、３．４５×１０~（－３）ｓ~（－１）;表现为失活与构象伸展变化基本平行。