C－反应蛋白与模型受体的特异性结合

Ｃ－反应蛋白是动物体内一种典型的急性期反应蛋白,本文人工合成了两种可以与Ｃ－反应蛋白特异性结合的兼性分子作为Ｃ－反应蛋白的模型受体,以便进一步在脂单层膜表面上组装Ｃ－反应蛋白的二维晶体。作为第一步工作。本文研究了兼性分子的特性以及荧光光谱方法监测兼性分子与Ｃ－反应蛋白之间特异性相互作用。荧光光谱实验结果表明受体与Ｃ－反应的特异结合会引起荧光强度的下降。     

铝与钙调蛋白相互作用的荧光光谱研究

本文研究了铝与钙调蛋白相互作用的荧光光谱。实验证明,Ａｌ＾３＋与ＣａＭ的结合所引起的构象变化与Ｃａ＾２＋与ＣａＭ结合所引起的构象变化既有相同之处,也有不同之处。Ａｌ＾３＋在ＣａＭ分子上的结合有特异性结合与非特异性结合两种情况。其特异性结合位点可能为２－３个,钙调蛋白的非竞争性拮抗剂酸枣仁皂甙Ａ（ＪｕＡ）可以继续抑制已被Ａｌ＾３＋部分抑制的ＰＤＥ－ＣａＭ的活力。     

CCR5的结构和功能：近两年的研究进展

ＣＣＲ５是趋化蛋白ＭＩＰ－１α,ＭＩＰ－１β、ＲＡＮＴＥＳ的特异性受体,主要表达于单核／巨细胞及Ｔ细胞膜上,具有Ｇ蛋白受体所特有的７个胯膜区。ＣＣＲ５与白细胞的趋经性,炎症反应,嗜Ｍφ型ＨＩＶ－１株对ＣＤ４细胞的感染密度相关。ＣＣＲ５的缺陷与个体对ＨＩＶ－１的抗性有关。本文就近两年在ＣＣＲ５结构特点,转录调控,信号传递,生物学功能等方面的研究进展作一综述。     

荧光共振能量转移效率的实时定量测量

荧光共振能量转移（FRET）广泛用于研究分子间的距离及其相互作用,与荧光显微镜结合,可定量获取有关生物活体内蛋白质、脂类、DNA和RNA的时空信息。随着绿色荧光蛋白（GFP）的发展,FRET荧光显微镜有可能实时测量活体细胞内分子的动态性质。提出了一种定量测量FRET效率以及供体与受体间距离的简单方法,仅需使用一组滤光片和测量一个比值,利用供体和受体的发射谱肖除光谱间的串扰。该方法简单快速,可实时定量测量FRET的效率和供体与受体间的距离,尤其适用于基于GFP的供体－受体对。     

胆碱脱氢酶光谱性质的研究

胆碱脱氢酶（ＣＤＨ）蛋白质部分内源荧光发射峰在３３５ｎｍ,并不受底物的影响,但底物可改变辅基ＦＡＤ部分的内源荧光光谱。应用ＦＴＩＲ技术研究了增溶ＣＤＨ的二级结构,其结果如下：５３．４％α－螺旋,２４．５％β－片层,１３．９％３１０－螺旋及０．５％β－回折。在ＣＤＨ处于非底物结合状态时,分子内部结构表现为α－螺旋以及β－片层优势构象,呈现出球状蛋白样的空间结构特征。在与底物作用过程中,３１０－螺旋的比例逐渐上升至４２％左右,与此同时α－螺旋结构则降低到３５％。提示了底物诱导ＣＤＨ分子内部发生了蛋白分子的重新折叠。     

IL－3、GM－CSF和IL－5受体及其信号传导

ＩＬ—３、ＧＭ－ＣＳＦ和ＩＬ—５是一类功能相似的造血因子,它们的受体至少包括配体特异性的α亚基和共用β亚基（βｃ）两部分。βｃ识别三因子的Ｎ端α－螺旋,而α亚基则识别三因子的Ｃ端α－螺旋,两者均为功能性的高亲和力受体所必需．由于βｃ胞内区较长,能结合Ｊａｂ和Ｓｔａｔ等信号分子,所以,三种因子受体受配体刺激后能诱导相似蛋白的酪氨酸磷酸化。另外,激酶的不同剪切型、转录因子的同分异构体、受体亚基的细胞型特异性以及信号传导通路的多重性等则为三因子作用特异性的基础。本文就对其主要内容作一综述。     

铝与钙调蛋白相互作用的荧光光谱研究

本文研究了铝与钙调蛋白相互作用的荧光光谱。实验证明,Al~3与CaM的结合所引起的构象变化与Ca~(2+)与CaM结合所引起的构象变化既有相同之处,也有不同之处。Al~(3+)在CaM分子上的结合有特异性结合与非特异性结合两种情况。其特异性结合位点可能为2—3个。钙调蛋白的非竞争性拮抗剂酸枣仁皂甙A(JuA)可以继续抑制已被Al~(3+)部分抑制的PDE-CaM的活力。     

胆碱脱氨酶光谱性质的研究

胆碱脱氢酶（ＣＤＨ）蛋白南部分内源荧光发射峰在３３５ｎｍ,并不受底物的影响,但底物可改变辅基ＦＡＤ部分的内源荧光光谱。应用ＦＴＩＲ技术研究了增溶ＣＤＨ的二级结构,其结果如下：５３．４％α螺旋,２４．５％β－片层,１３．９％３１０－螺旋及０．５％β回折。在ＣＤＨ处于非底物结合状态时,分子内部结构表现为α－螺旋以及β－片怪优势构象,呈现出球状蛋白样的空间结构特征,在与底物作用过程中,３１０－螺旋的比例     

牛心线粒体ATP酶抑制蛋白对酶的催化部位构象的影响

以标记在ＡＴＰ酶（Ｆ１）催化部位的ＴＮＰ－ＡＴＰ为荧光探针,比较测定了Ｆ１与其抑制蛋白（ＩＦ１）结合前后的ＴＮＰ－ＡＴＰ荧光光谱,荧光寿命和荧光偏振光谱,结果表明在ＩＦ１的作用下,酶分子催化部位的极性下降,ＴＮＰ－ＡＴＰ分子的自由度减小,提示ＩＦ１引起了Ｆ１催化部位的构象改变。     

R-藻蓝蛋白的分离及其结构表征

本文对传统藻胆蛋白的分离方法进行改进,利用柱层析法直接从新鲜多管藻提取分离出纯的Ｒ－藻蓝蛋白。分别用凝胶柱层析法和电泳法测定了其分子量。结果表明：通常实验条件下：Ｒ－藻蓝蛋白的最稳定聚集态是三聚体,其分子量为１２２．８ＫＤ,它由分子量分别为１８．１ＫＤ（α）和２０．５ＫＤ（β）两个亚基组成,其分子组成为（αβ）３。利用吸收和荧光光谱研究了Ｒ－藻蓝蛋白的光谱性质。Ｒ－ＰＣ的三聚体在可见光范围有两个明显的吸收峰,分别为５４６ｎｍ和６１４ｎｍ,与同系的Ｃ－ＰＣ和ＰＥＣ明显不同,表明各类色团光谱特性在三聚体状态基本不变,说明三聚体内色团间无强相互作用。Ｒ－ＰＣ荧光发射峰位于６４０ｎｍ,与同系的Ｃ－ＰＣ和ＰＥＣ基本一致,说明三聚体保证了高效的能量传递。单体的荧光发射呈现双峰（５６６ｎｍ,６４３ｎｍ）,说明单体内能量传递效率不高。从光谱性质可知,在ＰＣ家族中,Ｒ－ＰＣ具有最高的光能捕获效率     

Pentameric Two-Dimensional Crystallization of Rabbit C-Reactive Protein on Lipid Monolayers

As a member of the pentraxin family, C-reactive protein plays various roles in the nonspecific immunity of animals. Though soluble, C-reactive protein always functions on membranes. In order to study the structure of the membrane-bound protein and the reaction between protein and membranes, two-dimensional (2D) crystallization of rabbit C-reactive protein on lipid monolayers was performed. The 2D crystals composed of pentameric proteins were obtained on lipid monolayers by specific adsorption for the first time. The projection map at 26-A resolution is presented, which exhibits P2 symmetry with lattice parameters a = 158(+/-3) A, b = 92(+/-1) A, and gamma = 107(+/-1) degrees. The current work may give a basis for the further study on the structure of complexes made up of C-reactive protein with its functional binding molecules on membranes.     

Isolation and characterisation of goat C-reactive protein

A pentraxin was isolated from acute phase goat serum by its calcium-dependent affinity for agarose, and although it did not bind to phosphorylcholine immobilised on Sepharose, its binding to agarose was reversed by exposure to fluid phase phosphorylcholine. It was identified as goat C-reactive protein on the basis of its immunochemical cross-reactivity with human and bovine C-reactive protein. The molecule was composed of five identical, glycosylated, non-covalently associated subunits, each of molecular weight approx. 24,000. Acute phase serum levels in a small number of samples were not significantly different from normal levels (means 72 and 55 micrograms/ml, respectively), suggesting that goat C-reactive protein is not a major acute phase reactant. No other pentraxin was detected in goat serum.     

Studies of the interaction of RecA protein with DNA

Ethidium fluorescence assays were adapted for the rapid and sensitive detection of precA; in addition, fluorescence measurements on binding precA to linear, OC and CCC PM2 DNAs have enabled the stoichiometry of precA binding as well as the precA-induced unwinding angle of DNA to be determined. The stoichiometry of binding was independently confirmed by sedimentation analysis to be one precA molecule per 3 bp. The unwinding angle was also independently confirmed by measurements of fluorescence changes induced by the binding of precA to CCC DNA which was relaxed by topoisomerase to give a precA-induced unwinding angle of 51 degrees. Electron microscopy of OC DNA molecules which bound nonsaturating amounts of precA revealed that the length increase in DNA due to precA was approximately 55%. Finally, examination of negatively stained precA complexes with a variety of linear DNAs showed that the minor groove is the primary site of interaction for this protein.     

Effects of photoproducts on the binding properties of protoporphyrin IX to proteins

Photosensitisers are the photoactive molecules used in photodynamic therapy (PDT) of cancer. Despite the importance of their interaction with polypeptides, only the binding to plasma proteins has been investigated in some detail. In our study we compared the binding of Protoporphyrin IX (a clinically useful photosensitiser) to an immunoglobulin G, with the binding to albumins. Binding to IgG is relevant because a possible method of increasing tumour specificity of photosensitisers is to bind them to tumour-specific antibodies. Binding constants to albumins and the immunoglobulin were comparable ( congruent with6 x 10(-6) M(-1)). The apparent number of PPIX molecules bound to each protein was also within a similar range (from 4 to 7). The absence of a shift in the emission spectrum of PPIX bound to IgG, however, indicates that either larger aggregates of PPIX bind to the immunoglobulin or that the binding site leaves PPIX exposed to the buffer. We observed that PPIX photoproducts compete with PPIX for the same binding sites. The number of PPIX molecules bound to each protein in the presence of photoproducts decreased by 50-80%. Due to the spectral overlap between PPIX and its photoproducts, the binding in the presence of photoproducts was investigated using Derivative Synchronous Fluorescence Spectroscopy (DSFS) to improve the spectral separation between chromophores in solution. We also concluded that fluorescence measurements underestimate the number of PPIX molecules binding each protein. In fact, non-linear Scatchard plots (in the case of albumin binding) by definition yield a minimum number of molecules attached to a protein. Moreover, the binding of large aggregates, formed by an unknown number of PPIX molecules, to IgG results in the underestimate of the number of molecules bound. The number of PPIX molecules bound to these proteins is also much larger than the number of sites estimated by protein fluorescence quenching.     

Complement factor H binds to denatured rather than to native pentameric C-reactive protein

Binding of the complement regulatory protein, factor H, to C-reactive protein has been reported and implicated as the biological basis for association of the H402 polymorphic variant of factor H with macular degeneration. Published studies utilize solid-phase or fluid-phase binding assays to show that the factor H Y402 variant binds C-reactive protein more strongly than H402. Diminished binding of H402 variant to C-reactive protein in retinal drusen is posited to permit increased complement activation, driving inflammation and pathology. We used well validated native human C-reactive protein and pure factor H Y402H variants to test interactions. When factor H variants were incubated with C-reactive protein in the fluid phase at physiological concentrations, no association occurred. When C-reactive protein was immobilized on plastic, either non-specifically by adsorption in the presence of Ca(2+) to maintain its native fold and pentameric subunit assembly or by specific Ca(2+)-dependent binding to immobilized natural ligands, no specific binding of either factor H variant from the fluid phase was observed. In contrast, both factor H variants reproducibly bound to C-reactive protein immobilized in the absence of Ca(2+), conditions that destabilize the native fold and pentameric assembly. Both factor H variants strongly bound C-reactive protein that was denatured by heat treatment before immobilization, confirming interaction with denatured but not native C-reactive protein. We conclude that the reported binding of factor H to C-reactive protein results from denaturation of the C-reactive protein during immobilization. Differential binding to C-reactive protein, thus, does not explain association of the Y402H polymorphism with macular degeneration.     

HU binding to DNA: evidence for multiple complex formation and DNA bending

HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities.     

Thermal stability and ligand-binding properties of human plasma alpha 1-acid glycoprotein (orosomucoid) as determined with fluorescent probes

The fluorescence of 1,8-anilinonaphthalene sulfonate is enhanced and blue-shifted upon binding to alpha 1-acid glycoprotein, a human plasma protein of uncertain function. Fluorescence titrations of delipidated protein indicate at least two classes of binding sites having dissociation constants of 0.33 microM and 12 microM at 25 degrees C in 0.02 M potassium phosphate/0.15 M NaCl, pH 7.4. Exclusion chromatography measurements indicate only 1 binding site per mol protein, suggesting that the heterogeneity is due to differences between protein molecules, the origin of which remains unclear. The fluorescence of a mixture of dye and protein is progressively diminished upon addition of ethanol and other organic solvents whose presence could be detected at concentrations as low as 100 mM. Addition of the adrenergic drug propranolol to a mixture of alpha 1-acid glycoprotein (2.5 microM) and 1,8-anilinonaphthalene sulfonate (4 microM) caused a hyperbolic decrease in dye fluorescence to 30% of the initial value, with half-maximal response near 1 microM propranolol. When the protein-dye mixture was heated, the fluorescence of the dye exhibited a reversible downward transition with midpoint near 65 degrees C, compared to a midpoint of 58.5 degrees C obtained by intrinsic fluorescence in the absence of dye. This stabilization was confirmed with fluorescein-labeled protein, whose fluorescence polarization revealed a melting transition at 58.8 degrees C in the absence of ligands which increased by 5-6 Cdeg in the presence of 1,8-anilinonaphthalene sulfonate or propranolol. The sensitivity of 1,8-anilinonaphthalene sulfonate fluorescence to changes in the conformation and ligand environment of alpha 1-acid glycoprotein should facilitate efforts to understand the structure and function of this acute-phase reactant.     

1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain

The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0–7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ∼80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required.     

Secretion of C-reactive protein becomes more efficient during the course of the acute phase response

We studied the kinetics of synthesis and secretion of the acute phase plasma protein, C-reactive protein, in primary hepatocyte cultures prepared from rabbits manifesting differing degrees of the acute phase response to inflammatory stimulus. In cultures prepared from progressively more responsive animals, rate of C-reactive protein secretion increased to a much greater degree than did intracellular C-reactive protein content, resulting in a progressive decrease in the ratio of intracellular content to rate of secretion. This ratio, which represents the time required to secrete the amount of C-reactive protein contained within the intracellular pool, decreased from 18 h in cultures from unstimulated rabbits to 2.5 h in cells from highly responsive animals. In contrast, these ratios for albumin were short and fell within a narrow range (0.8-2.1 h). In pulse-chase labeling experiments, the time required for secretion of 50% of pulse-labeled C-reactive protein varied markedly, ranging from well over 6 h in cells from a minimally responsive animal to about 75 min in cells from a highly responsive rabbit. In contrast, the half-time for secretion of albumin was consistently about 45 min in the same cultures. Taken together, these findings indicate that the process by which C-reactive protein is secreted becomes more efficient during the course of the acute phase response. Recent studies have indicated that secretory proteins pass from the rough endoplasmic reticulum to Golgi at different and characteristic rates, possibly by a receptor-mediated process in which rate of transfer is determined by receptor affinity. We postulate that C-reactive protein secretion is regulated, during the course of the acute phase response, either by alterations in availability of specific receptors or by competition between different secretory proteins for a common receptor.