蛋白质相互作用的生物信息学研究进展

生命过程的分子基础在于生物分子之间的相互作用,其中蛋白质分子之间的相互作用占有极其重要的地位。研究蛋白质相互作用对于理解生命的真谛、探讨致病微生物的致病机理,以及研究新药提高人们的健康水平具有重要的作用。用生物信息学的方法研究蛋白质的相互作用已经取得了许多重要的成果,但也有很多问题还需解决。本文从蛋白质相互作用的数据库、预测方法、可预测蛋白质相互作用的网上服务、蛋白质相互作用网络等几方面,对蛋白质相互作用的生物信息学研究成果及其存在的问题做了概述。     

冬小麦不同生育期植株与土壤的硫素动态

S是植物生命活动必需元素之一,在植物细胞的结构和生理生化功能中都具有不可替代的作用.如在植物体中参于蛋白质合成、光合作用、呼吸作用、脂类合成、生物固氮、糖代谢等.     

蛋白质检测的方法学研究概述

蛋白质作为生命活动中起重要作用的生物大分子,与一切揭开生命奥秘的重大研究课题都有密切的关系.蛋白质的分离与检测是蛋白质研究的主要技术之一.我们就蛋白质检测的常规方法、电化学检测方法、分子生物学检测方法、电泳法和质谱法等进行了简要综述.     

植物E3泛素连接酶的分类与功能

蛋白质泛素化作为一种重要的翻译后修饰,通过介导特定蛋白质的降解,广泛地参与到植物生长发育、胁迫响应、信号转导等一系列生命活动过程中,在植物的生命周期中具有重要意义。E3泛素连接酶能够特异性地识别靶蛋白,在泛素化途径中起决定性作用。因此,研究植物E3泛素连接酶的功能及其作用机理具有重要的意义。该文介绍了目前E3泛素连接酶分类与功能方面的研究进展,为深入探讨E3泛素连接酶在植物生命活动过程中的调控机制提供借鉴。     

用亲和纯化方法筛选与gankyrin相互作用的蛋白质

目的：作为一个新发现的癌蛋白,gankyrin在肝癌细胞的发生和形成中发挥重要作用。筛选与gankyrin相互作用的蛋白质,从而进一步探讨gankyrin的作用机制。方法：采用亲和纯化技术及质谱鉴定筛选与gankyrin相互作用的蛋白质。结果：初步筛选出了6个与gankyrin发生相互作用的蛋白质,经与MSDB或NCBInr数据库比对,这6个蛋白质均是26S蛋白酶体的组成部分,其中proteasome（prosome,macropain）26S subunit,ATPase,4为已报道的蛋白质,证实结果的可靠性。结论：采用亲和纯化的方法可以有效地筛选出与gankyrin相互作用的蛋白质,为进一步研究gankyrin的作用机制及生物学功能奠定了基础。     

蛋白质的化学改性及其应用

蛋白质是生命活动的重要物质之一。蛋白质种类繁多,各具有自己的特殊的生物功能,如催化、传递系统,调节代谢过程和参与结构成分等等。蛋白质功能的多样性是与其化学结构以及由化学结构所决定的空间结构的多样性分不开的。从分子水平上来说,许多蛋白质作用的对象是小分子,如酶与底物或抑制剂间的作用,抗体与抗原尤其是半抗原间的作用。这些底物或     

细菌中常见的蛋白翻译后修饰

蛋白质的翻译后修饰在生物体生命活动中发挥着重要作用,大部分蛋白质都会经历翻译后修饰。对这些修饰的了解和掌握非常重要,因为这些修饰可能会改变蛋白质的物理及化学性质,如折叠、构象、稳定性及活性,从而改变蛋白的功能。此外,修饰基团本身也可能具有某些功能。因此,分析研究蛋白质翻译后修饰具有重要意义。细菌中常见的翻译后修饰过程有糖基化、磷酸化和乙酰化,我们简要综述了这几种修饰过程。     

蛋白质结构和功能关系的教学实例——绿色荧光蛋白的发现和发展

蛋白质是生命的物质基础,几乎参与生命活动的每一个过程。蛋白质功能也存在多样性,如生物催化、代谢调节、免疫保护、转运储存等作用,这些功能的发挥都与蛋白的结构密切相关。因此,“蛋白质的结构与功能”是生物化学课程中的重要章节,涉及到生物学、化学、分子生物学等多学科的交叉,具有理论抽象和知识点繁杂等特点。本文以“绿色荧光蛋白的发现和发展”为例,探讨了蛋白质结构和功能关系教学的具体设计和实践,希望使“蛋白质的结构与功能”的课程教育更加简明易懂,并提高学生对课堂内容的重视度,旨在为生物化学教学改革提供参考。     

原核生物的蛋白质翻译后修饰

蛋白质翻译后修饰是调节蛋白质生物学功能的关键步骤之一,是蛋白质动态反应和相互作用的一个重要分子基础,同时,它也是细胞信号网络调控的重要靶点.目前,蛋白质翻译后修饰已经成为国际上蛋白质研究的一个极其重要的热点.在原核生物生命活动中,蛋白质的翻译后修饰具有十分重要的作用,如参与细胞信号传导、物质的代谢、蛋白质的降解、致病微生物的致病过程等.综述了经典原核生物蛋白质翻译后修饰的种类、机制和功能,同时介绍了最近发现的原核生物的全局性乙酰化修饰以及结核分枝杆菌中类泛素化修饰.     

探讨酶的记忆

酶是否具有对其反应底物形成记忆是一个值得探讨的问题。在底物与酶相互作用中,底物诱导酶产生与其互补结合的构象,其构象的稳定性,决定了酶对底物的高度选择性。尽管某种酶能够与多个底物Sn(n=1,2,3…)发生反应,但如果酶先与S1反应,则酶将降低对S2、S3等底物的选择性,反之亦然。这似乎是酶(蛋白质)与底物S1作用后,对S1形成了记忆。     

Distinct functions of elongation factor G in ribosome recycling and translocation

Elongation factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, together with ribosome recycling factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is strongly inhibited by vanadate, an analog of inorganic phosphate (Pi), indicating that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in single-round translocation is not affected by vanadate, while the turnover reaction is strongly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suggesting that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recycling. Our results indicate that conformational coupling between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tight coupling between the conformational change of EF-G induced by Pi release and ribosome disassembly.     

核糖体展示口蹄疫单链抗体库的构建

目的:构建库容量大、多样性好的核糖体展示口蹄疫单链抗体(scFv)库。方法: 分离口蹄疫病毒免疫的兔脾细胞,提取总RNA,用RT-PCR扩增兔抗体的重链可变区(VH)基因和轻链可变区(VL)基因,同时扩增作为间隔区的兔抗体Ck基因;采用重叠延伸PCR (简称SOE-PCR)技术连接VH-VL基因,同时引入T7启动子和核糖体结合位点序列,体外构建核糖体展示scFv库模板,连接pMD18-T载体转化E.coli DH5α大肠杆菌,挑取阳性克隆测序以鉴定scFv组装。结果:成功构建了库容量达8.21×10¹³的兔源口蹄疫核糖体展示scFv库。结论: 构建的大容量兔源性口蹄疫核糖体展示抗体库可以成为进一步筛选特异性口蹄疫单链抗体的实验平台,为开发诊断性口蹄疫单链抗体奠定了很好的实验基础。     

The ribosome as a molecular machine: the mechanism of tRNA-mRNA movement in translocation

Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.     

On the rate of ribosome translocation anisotropy and ribosome saturation in the polysome

This study examines the rate of ribosome translocation in the mammalian polysome engaged in protein synthesis by utilizing our knowledge of the hydrodynamic behavior of the rat liver polysomes, sedimenting in a linear sucrose density gradient. The average distance between adjacent ribosomes in the polysome was estimated assuming an extended linear configuration of the polysomes during sedimentation. Based on this estimate, the velocity of ribosome movement along the messenger RNA appears to be non-uniform and inversely related to the ribosome content of the polysome. Such non-uniformity prevails at stages of translation prior to ribosome “saturation” of the polysome. A correlation has been made between the results reported herein and previously published evidence on the rate of polypeptide chain synthesis. The steady-state condition for the polypeptide chain assembly is viewed as representing the state of ribosome “saturation”, characterized by a minimal ribosome velocity and a maximum density of ribosome distribution, both functions being uniform throughout the entire length of the polysome.     

Structural basis for aminoglycoside inhibition of bacterial ribosome recycling

Aminoglycosides are widely used antibiotics that cause messenger RNA decoding errors, block mRNA and transfer RNA translocation, and inhibit ribosome recycling. Ribosome recycling follows the termination of protein synthesis and is aided by ribosome recycling factor (RRF) in bacteria. The molecular mechanism by which aminoglycosides inhibit ribosome recycling is unknown. Here we show in X-ray crystal structures of the Escherichia coli 70S ribosome that RRF binding causes RNA helix H69 of the large ribosomal subunit, which is crucial for subunit association, to swing away from the subunit interface. Aminoglycosides bind to H69 and completely restore the contacts between ribosomal subunits that are disrupted by RRF. These results provide a structural explanation for aminoglycoside inhibition of ribosome recycling.     

An analysis of ribosomal protein during the development of Xenopus laevis

Summary Conditions for the isolation and purification of ribosome proteins from developing Xenopus embryos have been established. The procedure involves the preparation of ribosome gradients, and from the monosomes and polysomes their protein. These proteins are purified by an ammonium chloride wash and are separated by electrophoresis. Results indicate that differences do not exist between monosome ribosome proteins from different developmental stages, but do exist between these monosomes and ribosomal protein from postgastrula polysomes. The possible role of the ribosome in translation-level control is discussed.     

Structural changes in the ribosome during the elongation cycle

Ample data on structural changes that arise in the ribosome during translation have been accumulated. The most interesting information on such changes has been obtained by cryoelectron microscopy of ribosome complexes with various ligands and by rRNA site-directed mutagenesis combined with a structural analysis of the ribosome by a chemical modification technique (chemical probing). The review considers the best-known structural changes that arise in the translating ribosome upon its interactions with tRNA and the elongation factors. The changes are discussed in the context of interactions between the functional centers of the ribosome. A universal system of rRNA helices and proteins is described in detail. The system integrates the functional centers of the ribosome and allows transduction of allosteric conformational signals. Biochemical data are considered in terms of the structures and interactions of ribosomal elements, and a hypothesis is advanced that the position of the GTPase-associated center in the ribosome regulates the binding of the elongation factors.     

RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation

Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.     

Structure of the mammalian 80S ribosome at 8.7 A resolution

In this paper, we present a structure of the mammalian ribosome determined at approximately 8.7 A resolution by electron cryomicroscopy and single-particle methods. A model of the ribosome was created by docking homology models of subunit rRNAs and conserved proteins into the density map. We then modeled expansion segments in the subunit rRNAs and found unclaimed density for approximately 20 proteins. In general, many conserved proteins and novel proteins interact with expansion segments to form an integrated framework that may stabilize the mature ribosome. Our structure provides a snapshot of the mammalian ribosome at the beginning of translation and lends support to current models in which large movements of the small subunit and L1 stalk occur during tRNA translocation. Finally, details are presented for intersubunit bridges that are specific to the eukaryotic ribosome. We suggest that these bridges may help reset the conformation of the ribosome to prepare for the next cycle of chain elongation.