蛋白质间相互作用的研究策略

病毒感染肝细胞后,病毒的核酸、蛋白与肝细胞的核酸、蛋白等生物大分子之间的相互作用是病毒致病的主要分子机制之一。研究蛋白的功能,常常通过改变蛋白的表达水平,观察细胞的生物学特性的变化,来研究蛋白相应的生物学功能。近年来,基因和蛋白质结构的分子生物学技术与计算机分析技术结合起来,形成了目前极具潜力的新兴交叉学科-生物信息学技术。     

心血管系统蛋白质组的研究进展

继基因组之后蛋白质组研究已成为当前生物医学研究的热点，检查蛋白质组的变化可深入理解基因组分析不能明确的细胞和分子机制，蛋白质组技术的发展促进了心血管疾病在分子机制上更全面的研究。本文概述了蛋白质组技术及其在心血管系统的研究进展。     

酵母双杂交系统在痘病毒与宿主相互作用中的研究进展

痘病毒全基因组序列分析为痘病毒的研究提供了丰富的信息。近年来,一些痘病毒编码的蛋白之间,以及病毒蛋白与宿主蛋白之间的相互作用的研究证明其在痘病毒复制、感染、致病及传播过程中起关键作用,因此受到了广泛的关注。酵母双杂交系统是研究蛋白质之间相互作用的重要工具,该技术可以发现新蛋白之间的相互作用,确定蛋白相互作用的区域,从而为研究各蛋白质的生物学功能以及蛋白质之间相互作用的分子机制奠定基础。本文综述了酵母双杂交系统在痘病毒蛋白之间及病毒-宿主相互作用的研究现状及优缺点。     

蛋白质组技术在研究肺癌发病机制中的作用

蛋白质组是在一种细胞内存在的全部蛋白质，功能蛋白质组指细胞内与某个功能有关或在某种条件下的一群蛋白质。蛋白质组学是以蛋白质组为研究对象的新的研究领域，它从整体水平研究细胞内动态变化的蛋白质组成成分、表达水平与修饰状态，了解蛋白质之间的相互作用与联系，解释蛋白质功能与细胞生命活动的规律。蛋白质组技术的应用为研究肺癌的发生、发展机制提供了新的手段。     

心血管系统蛋白质组的研究进展

继基因组之后蛋白质组研究已成为当前生物医学研究的热点,检查蛋白质组的变化可深入理解基因组分析不能明确的细胞和分子机制,蛋白质组技术的发展促进了心血管疾病在分子机制上更全面的研究.本文概述了蛋白质组技术及其在心血管系统的研究进展.     

疾病蛋白质组研究进展

蛋白质组是继人类基因组计划之后，生物医学领域的又一研究热点，随着蛋白质组研究技术的日趋完善，蛋白质组研究已开始从建立数据库走向解决生命科学的重大问题，在肿瘤和神经系统疾病的发生与诊断以及病理研究等方面已经得到重大。疾病蛋白质组的深入研究，将对重大疾病的发病机制、疾病诊断、疾病预防和治疗提供重要的理论基础。     

蛋白质翻译后修饰及其生物学功能研究进展

<正>后基因组时代的到来意味着生命科学研究重心转向功能基因组学及功能蛋白质组学等新领域。蛋白质翻译后修饰是蛋白质组学的重要组成部分。蛋白质经翻译后修饰改变自身的空间构象、活性、稳定性及其与其他分子相互作用等方面的性能,从而参与调节机体多样化的生命活动。多数蛋白质存在翻译后修饰,目前已知的蛋白质共价修饰方式多达200余种,主要包括磷酸化、亚硝基化、硝基化、泛素化和小泛素相关修饰物化(SUMO)等。我们就蛋白质翻译后修     

蛋白质芯片激光解析电离技术在肺癌研究中的应用

蛋白质芯片表面加强激光解析电离-飞行时间-质谱(SELDI-TOF-MS)技术是蛋白质组学研究的全新技术平台,进一步提高了蛋白质分离和鉴定的速度。并且在肺肿瘤生物标志物筛选、鉴别肺癌抗原和蛋白质指纹图谱方面取得突破性进展。今后该技术在肿瘤蛋白质组学研究中有更加广阔的应用前景。     

蛋白质组技术在消化系肿瘤研究的进展

随着人类基刚组计划的完成，后基因组时代即蛋白质组的研究已拉开了序幕。蛋白质组技术的应用有望对肿瘤学的研究，包括消化系肿瘤的研究提供新的手段，可以进一步阐明其发生发展的机制。本文对蛋白质组研究的相关技术进行了介绍，并概述了消化系肿瘤中蛋白质组技术的研究进展。     

抗体基因芯片应用于检测鼻咽癌组织切片蛋白质表达谱的研究

目的研究应用抗体基因芯片检测鼻咽癌组织特异性蛋白质表达谱的新技术方案。方法利用噬菌体抗体库筛选出抗鼻咽癌的特异性抗体库,以扩增抗体基因V-D-J片段作为标识分子用于制备抗体基因芯片;通过特异性抗体库与鼻咽癌肿瘤组织结合后,使标记引物扩增的V-D-J序列与抗体基因芯片进行杂交后显示癌组织的蛋白质表达谱,从而选择性通过免疫组织化学显示其中关键性蛋白质原位表达状况。结果肿瘤组织结合的抗体滴度可达到106～108数量级,标记的DNA分子效价约为10-6～10-8,以该文一次检测20个抗体为例,基因芯片检出有10～14个阳性蛋白质表达谱;免疫组织化学可显示阳性抗体在细胞中的结合部位。结论以抗体基因芯片为核心的技术方案,能在组织切片上同时检测含多个蛋白质表达谱,可以广泛应用于基础研究和临床诊疗过程。     

New Technologies: Bioluminescence Resonance Energy Transfer (BRET) for the Detection of Real Time Interactions Involving G-Protein Coupled Receptors

The natural phenomenon of bioluminescence resonance energy transfer (BRET) has become an extremely useful tool for studying protein-protein interactions in the laboratory, including those involving G-protein coupled receptors (GPCRs). The technology involves fusion of donor and acceptor molecules to proteins of interest. Following assessment to ensure correct functionality, co-expression of fusion constructs in live cells enables their interaction to be studied in real time in a quantitative manner. Energy is transferred from the donor to the acceptor when in close proximity, resulting in fluorescence emission at a characteristic wavelength. The energy emitted by the acceptor relative to that emitted by the donor is termed the BRET signal. It is dependent upon the spectral properties, ratio, distance and relative orientation of the donor and acceptor molecules, as well as the strength and stability of the interaction between the proteins of interest. The ability to study interactions in live mammalian cells circumvents many of the problems associated with techniques such as co-immunoprecipitation and yeast two-hybrid screening. Furthermore, the high sensitivity of BRET enables the study of proteins at physiological concentrations, a significant advantage over techniques that require high levels of protein expression. BRET technology has already made a substantial contribution to our understanding of GPCRs and protein-protein interactions, in particular by providing strong evidence that GPCRs homo- and hetero-oligomerize. New BRET detection systems and the potential for novel high throughput screening applications means that BRET promises to play an important role in future research and drug discovery.     

Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism

Fluorescence resonance energy transfer and fluorescence polarization anisotropy are used to investigate single molecules of the enzyme staphylococcal nuclease. Intramolecular fluorescence resonance energy transfer and fluorescence polarization anisotropy measurements of fluorescently labeled staphylococcal nuclease molecules reveal distinct patterns of fluctuations that may be attributed to protein conformational dynamics on the millisecond time scale. Intermolecular fluorescence resonance energy transfer measurements provide information about the dynamic interactions of staphylococcal nuclease with single substrate molecules. The experimental methods demonstrated here should prove generally useful in studies of protein folding and enzyme catalysis at single-molecule resolution.     

哺乳动物血红素降解途径中蛋白质的相互作用研究进展

血红素加氧酶(HO)通过细胞色素P450还原酶(CPR)从NADPH传递电子,催化血红素降解为胆绿素、CO和铁的限速步骤。然后胆绿素还原酶(BVR)催化胆绿素转化为胆红素。目前已用突变结合动力学、光谱(荧光和核磁共振)、表面等离子体共振、交联、凝胶过滤和分析超速离心研究等评估血红素加氧酶-2(HO-2)与细胞色素P450还原酶(CPR)、胆绿素还原酶(BVR)的相互作用。本文介绍哺乳动物血红素降解途径中蛋白质的相互作用的研究进展。     

The kinetic basis of peptide exchange catalysis by HLA-DM

The mechanism by which the peptide exchange factor HLA-DM catalyzes peptide loading onto structurally homologous class II MHC proteins is an outstanding problem in antigen presentation. The peptide-loading reaction of class II MHC proteins is complex and includes conformational changes in both empty and peptide-bound forms in addition to a bimolecular binding step. By using a fluorescence energy transfer assay to follow the kinetics of peptide binding to the human class II MHC protein HLA-DR1, we find that HLA-DM catalyzes peptide exchange by facilitating a conformational change in the peptide-bound complex, and not by promoting the bimolecular MHC-peptide reaction or the conversion between peptide-receptive and -averse forms of the empty protein. Thus, HLA-DM serves essentially as a protein-folding or conformational catalyst.     

Use of chimeric fluorescent proteins and fluorescence resonance energy transfer to monitor cellular responses

In recent years, the development of new technologies based on the green fluorescent protein and fluorescence resonance energy transfer has introduced a new perspective in the study of cell biology. Real-time imaging of fluorescent biosensors has made it possible to directly visualize individual molecular events as they happen in intact, live cells, providing important and original insights for our understanding of biologically relevant problems. This review discusses some essential methodological aspects concerning the generation and use of fluorescence resonance energy transfer-based biosensors and presents selected examples of specific applications that highlight the power of this technology.     

Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric G proteins (Galphabetagamma). Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with Galphabetagamma. To investigate whether a G protein-coupled receptor monomer can activate Galphabetagamma, we used the retinal photoreceptor rhodopsin and its cognate G protein transducin (G(t)) to determine the stoichiometry of rhodopsin/G(t) binding and the rate of catalyzed nucleotide exchange in G(t). Purified rhodopsin was prepared in dodecyl maltoside detergent solution. Rhodopsin was monomeric as concluded from fluorescence resonance energy transfer, copurification studies with fluorescent labeled and unlabeled rhodopsin, size exclusion chromatography, and multiangle laser light scattering. A 1:1 complex between light-activated rhodopsin and G(t) was found in the elution profiles, and one molecule of GDP was released upon complex formation. Analysis of the speed of catalytic rhodopsin/G(t) interaction yielded a maximum of approximately 50 G(t) molecules per second and molecule of activated rhodopsin. The bimolecular rate constant is close to the diffusion limit in the diluted system. The results show that the interaction of G(t) with an activated rhodopsin monomer is sufficient for fully functional G(t) activation. Although the activation rate in solution is at the physically possible limit, the rate in the native membrane is still 10-fold higher. This is likely attributable to the precise orientation of the G protein to the membrane surface, which enables a fast docking process preceding the actual activation step. Whether docking in membranes involves the formation of rhodopsin dimers or oligomers remains to be elucidated.     

Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids

We have found that the efficiency of fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to the 5′ ends of a DNA duplex is significantly affected by the relative orientation of the two fluorophores. The cyanine fluorophores are predominantly stacked on the ends of the helix in the manner of an additional base pair, and thus their relative orientation depends on the length of the helix. Observed fluorescence resonance energy transfer (FRET) efficiency depends on the length of the helix, as well as its helical periodicity. By changing the helical geometry from B form double-stranded DNA to A form hybrid RNA/DNA, a marked phase shift occurs in the modulation of FRET efficiency with helix length. Both curves are well explained by the standard geometry of B and A form helices. The observed modulation for both polymers is less than that calculated for a fully rigid attachment of the fluorophores. However, a model involving lateral mobility of the fluorophores on the ends of the helix explains the observed experimental data. This has been further modified to take account of a minor fraction of unstacked fluorophore observed by fluorescent lifetime measurements. Our data unequivocally establish that Förster transfer obeys the orientation dependence as expected for a dipole–dipole interaction.     

Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I.

We have devised a microspectroscopic strategy for assessing the intracellular (re)distribution and the integrity of the primary structure of proteins involved in signal transduction. The purified proteins are fluorescent-labeled in vitro and reintroduced into the living cell. The localization and molecular state of fluorescent-labeled protein kinase C beta I isozyme were assessed by a combination of quantitative confocal laser scanning microscopy, fluorescence lifetime imaging microscopy, and novel determinations of fluorescence resonance energy transfer based on photobleaching digital imaging microscopy. The intensity and fluorescence resonance energy transfer efficiency images demonstrate the rapid nuclear translocation and ensuing fragmentation of protein kinase C beta I in BALB/c3T3 fibroblasts upon phorbol ester stimulation, and suggest distinct, compartmentalized roles for the regulatory and catalytic fragments.