SMR蛋白:一种跨膜蛋白演化研究的模式蛋白

多药抗性小蛋白(small multidrug resistance protein,SMR)是一类由4个跨膜螺旋束组成的跨膜质子梯度驱动的内膜转运蛋白,一般由100~140个氨基酸残基组成,结构相对简单,在特定去垢剂中相对稳定并具有功能活性,是研究细菌的耐药机制、跨膜转运蛋白的结构功能以及膜蛋白演化的一类很好的模式蛋白家族。同时,由于其灵活多变的拓扑学分布以及在膜转运蛋白演化中节点的位置,成为研究和追寻膜蛋白演化路径的理想的模式蛋白。现分别从SMR蛋白家族的系统发生分布与进化、SMR蛋白的耐药性与转运机制、SMR蛋白的结构等3个方面对本领域的研究近况进行分析和总结。     

去垢剂在膜蛋白研究中的应用

去垢剂是同时具有亲水极性基团和疏水非极性基团的双极性分子,能够使脂膜解体释放膜蛋白,并在溶液中为去膜状态下的膜蛋白提供疏水环境,维持和保护膜蛋白的疏水跨膜结构,在膜蛋白的结构和功能研究中有重要的意义。去垢剂的双极性和理化特性,如临界胶束浓度能够极大影响去垢剂和膜蛋白间的相互作用。在膜蛋白研究中,需要充分利用去垢剂的结构和特性:一方面,需要利用去垢剂代替脂质分子支持和稳定去膜状态下膜蛋白的结构和功能;另一方面,需要控制去垢剂和膜蛋白的相互作用,以满足膜蛋白结构研究如蛋白质结晶试验的要求。简要介绍了去垢剂在膜蛋白研究中的最新应用进展,涉及去垢剂在膜蛋白离体表达、分离和纯化、以及结构研究中的应用。     

膜蛋白的拓扑学

膜蛋白的拓扑学是研究膜蛋白三维结构的出发点．利用融合蛋白和化学修饰等实验技术已确定了很多膜蛋白的拓扑学．对膜蛋白的转运与插膜的研究确定可能存在两类插膜元件．对已知拓扑学的膜蛋白的统计分析以及蛋白质工程的研究表明存在膜蛋白拓扑学的内正规则．目前已形成预测膜蛋白的拓扑学的比较可靠的策略,这在反向生物学上具有重要意义.但要进行三维结构的预测还有许多路要走．     

铜绿假单胞菌Psl多糖转运蛋白PslD的表达与纯化研究

条件性致病菌铜绿假单胞菌是细菌生物被膜研究的模式菌,其分泌的胞外多糖Psl在生物被膜形成中起关键作用。PslD为Psl多糖的转运蛋白,由256个氨基酸构成,生物信息学分析揭示其有N端信号肽且为一次跨膜蛋白。分离纯化完整跨膜蛋白需要去垢剂的作用,去垢剂种类繁多且性质不一,研究设计了一套筛选溶解PslD的去垢剂的方案。通过抗组氨酸标签的Western blot分析,n-Decyl-β-D-maltopyranoside (DM),n-Decyl-N,N-dimethylamine-N-oxide(DDAO)及n-Dodecyl-N,N-dimethylamine-N-oxide(LDAO)被认为溶解PslD的效率较高。通过改变总蛋白与去垢剂比例,进一步优化了去垢剂的溶解条件即8 mg/mL总蛋白:质量分数为1%的 LDAO。在此溶解条件下,仅通过第一步Ni柱亲和纯化目的蛋白纯度可到达80%以上,这为进一步结晶尝试及其结构生物学研究奠定基础。     

叶绿体蛋白质组研究进展

亚细胞蛋白质组学是近年来蛋白组学研究中的一个热点。通过细胞器的纯化和亚细胞组分的分离,降低了样品的复杂性,增大了相应蛋白质组分的富集,有利于由此分离获得的蛋白质的序列分析及功能鉴定。叶绿体蛋白质组为植物亚细胞蛋白质组学研究中相对全面的一部分,利用亚细胞分离结合双向电泳技术系统地鉴定叶绿体中蛋白质组分是获取叶绿体蛋白质信息、确定其功能的重要技术手段。本文就近年来植物叶绿体蛋白质组涵盖的叶绿体内、外被膜、叶绿体基质、类囊体膜和类囊体腔蛋白的研究进行综述,以全面认识叶绿体蛋白的组成、特点及其在叶绿体生理生化代谢网络中的作用。     

结核分枝杆菌膜蛋白的异源表达与纯化研究进展

膜蛋白是一类与生物膜相互作用、具有重要功能和独特结构的蛋白质。异源表达纯化一直是了解膜蛋白结构和功能的重要瓶颈。结核分枝杆菌作为典型的胞内致病菌,其膜蛋白的研究具有很好的代表性以及重要意义。目前用于表达膜蛋白的有大肠杆菌、酵母、哺乳动物细胞等表达系统,但结核菌膜蛋白的表达宿主还往往局限于大肠杆菌。异源表达需要综合考虑蛋白的来源、疏水性、跨膜区等特性。低温、加入共表达因子以及改变培养条件有助于结核菌膜蛋白的可溶性表达。另外,包涵体复性也是获得结核菌目的膜蛋白的重要途径。随着新的表达系统,新的促可溶表达策略,新的包涵体复性手段,新的纯化方法的应用,将有更多的膜蛋白异源表达纯化成功,为蛋白质功能研究奠定基础。     

蛋白质组学技术在病毒学研究中的应用

蛋白质组学是门研究细胞和组织中蛋白质的存在及其结构、翻译后修饰、表达水平,最终阐述蛋白质群体功能的学科[1],它不仅是对机体产生的所有蛋白进行系统分离解析,更重要的是对这些蛋白质如何行使功能作出解释.利用先进的蛋白质组学技术研究病毒及相闰关宿主蛋白的表达、翻译后修饰、定位、相互作用和功能进行系统研究,有助于深入了解病素毒复复制、与宿主相互作用以及引发疾病的机制。     

短胶法预处理含高浓度去垢剂的蛋白样品

蛋白质组学研究中常使用含高浓度去垢剂的缓冲液以提高蛋白提取效率,但这种缓冲液会对下游蛋白质定量、酶解、质谱分析等步骤造成干扰。短胶法可用于含高浓度去垢剂样品的预处理,文中进一步评价了短胶法(Short gel)用于含高去垢剂的蛋白样品定量和预处理的效果。使用牛血清白蛋白作为标准蛋白分析了短胶法定量的线性范围,结果发现短胶法定量的线性范围为1-8μg,提示可对这一范围内的样品蛋白进行定量检测。对标准蛋白定量的线性度达到0.999,且重复性好,不容易受到蛋白表达模式变化的影响,结果可靠。短胶法定量的蛋白可通过胶内酶解,直接进行液相色谱-质谱联用(Liquid chromatography-tandem mass spectrometry,LC-MS/MS)分析,质谱信号较好。结果提示直接用短胶法预处理含高浓度去垢剂的蛋白样品,值得在蛋白质组学研究中推广。     

龙须菜叶绿素-蛋白复合物的分离及鉴定

以海洋经济海藻--龙须菜(Gracilarialemaneiformis)为材料,机械破碎与超声波相结合破碎这种含胶量非常多的细胞,蔗糖密度梯度超速离心纯化其类囊体膜,去垢剂Triton X-100增溶纯化的类囊体膜,再用蔗糖密度梯度超速离心方法分离其叶绿素-蛋白质复合物.P700差示光谱鉴定分离的光系统Ⅰ(PSⅠ)颗粒,并且检测到了具有DCIP光还原活性的光系统Ⅱ(PSⅡ)颗粒.结果表明,尽管海藻类囊体膜增溶困难,但只要条件合适,可以得到具有活性的光系统颗粒.     

蛋白质组研究中细胞质膜的纯化和纯度鉴定研究进展

细胞质膜是构成细胞对外界环境的屏障和细胞内外环境交流的界面,镶嵌或连接于其中的蛋白质参与细胞/细胞以及细胞/细胞外基质的识别、信号的接受和跨膜传导、细胞内外物质的转运;此外,质膜蛋白质在药物研发中也起着非常重要的作用,在现有的药靶中质膜蛋白质占70%.因此质膜蛋白质组学研究成为亚细胞蛋白质组学研究的热点.然而在质膜蛋白质组学的研究中,由于很难获得高纯度的质膜样品,因此这一领域的研究具有很大的挑战性.现主要对质膜及其微区结构的纯化方法和质膜纯度的评价标准作扼要的介绍.     

Collection and characterisation of bacterial membrane proteins

A general strategy for the amplified expression in Escherichia coli of membrane transport and receptor proteins from other bacteria is described. As an illustration we report the cloning of the putative alpha-ketoglutarate membrane transport gene from the genome of Helicobacter pylori, overexpression of the protein tagged with RGS(His)6 at the C-terminus, and its purification in mg quantities. The retention of structural and functional integrity was verified by circular dichroism spectroscopy and reconstitution of transport activity. This strategy for overexpression and purification is extended to additional membrane proteins from H. pylori and from other bacteria.     

α-螺旋跨膜蛋白拓扑结构预测方法的评价

在基因组数据中,有20%～30%的产物被预测为跨膜蛋白,本文通过对膜蛋白拓扑结构预测方法进行分析,并评价其结果,为选择更合适的拓扑结构预测方法预测膜蛋白结构。通过对目前已有的拓扑结构预测方法的评价分析,可以为我们在实际工作中提供重要的参考。比如对一个未知拓扑结构的跨膜蛋白序列,我们可以先进行是否含有信号肽的预测,参考Polyphobius和SignalP两种方法,若两种方法预测结果不一致,综合上述对两种方法的评价,Polyphobius预测的综合能力较好,可取其预测的结果,一旦确定含有信号肽,则N端必然位于膜外侧。然后结合序列的长度,判断蛋白是单跨膜还是多重跨膜,即可参照上述评价结果,选择合适的拓扑结构预测方法进行预测。     

Production of membrane proteins using cell–free expression systems

Different overexpression systems are widely used in the laboratory to produce proteins in a reasonable amount for functional and structural studies. However, to optimize these systems without modifying the cellular functions of the living organism remains a challenging task. Cell-free expression systems have become a convenient method for the high-throughput expression of recombinant proteins, and great effort has been focused on generating high yields of proteins. Furthermore, these systems represent an attractive alternative for producing difficult-to-express proteins, such as membrane proteins. In this review, we highlight the recent improvements of these cell-free expression systems and their direct applications in the fields of membrane proteins production, protein therapy and modern proteomics.     

A systematic assessment of mature MBP in membrane protein production: overexpression, membrane targeting and purification

Obtaining enough membrane protein in native or native-like status is still a challenge in membrane protein structure biology. Maltose binding protein (MBP) has been widely used as a fusion partner in improving membrane protein production. In the present work, a systematic assessment on the application of mature MBP (mMBP) for membrane protein overexpression and purification was performed on 42 membrane proteins, most of which showed no or poor expression level in membrane fraction fused with an N-terminal Histag. It was found that most of the small membrane proteins were overexpressed in the native membrane of Escherichia coli when using mMBP. In addition, the proteolysis of the fusions were performed on the membrane without solubilization with detergents, leading to the development of an efficient protocol to directly purify the target membrane proteins from the membrane fraction through a one-step affinity chromatography. Our results indicated that mMBP is an excellent fusion partner for overexpression, membrane targeting and purification of small membrane proteins. The present expression and purification method may be a good solution for the large scale preparation of small membrane proteins in structural and functional studies.     

Bacterial membrane proteomics

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.     

Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies

Despite recent successes in the structure determination of eukaryotic membrane proteins, the total number of structures of these important proteins is severely underrepresented in the Protein Data Bank. Although prokaryotic homologues provide valuable mechanistic insight, they often lack crucial details, such as post-translational modification and additional intra or extracellular domains that are important for understanding the function and regulation of these proteins in eukaryotic cells. The production of milligram quantities of recombinant protein is still a serious obstacle to the structural and functional characterization of these proteins. Here, we report a modification to a previously described over expression system using the simple eukaryote Saccharomyces cerevisiae that can increase overall protein yield and improve downstream purification procedures. Using a metabolic marker under the control of a truncated promoter, we show that expression levels for several membrane transporters are increased fourfold. We further demonstrate that the increase in expression for our test proteins resulted in a concomitant increase in functional protein. Using this system, we were able to increase the expression level of a plant transporter, NRT1.1, which was a key factor in its structural and functional characterization.     

A novel and simple method of production and biophysical characterization of a mini-membrane protein, Ost4p: a subunit of yeast oligosaccharyl transferase

Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction. In eukaryotes, oligosaccharyl transferase (OT), a multi-subunit membrane-associated enzyme complex, catalyzes this reaction in newly synthesized proteins. In Saccharomyces cerevisiae, OT consists of nine nonidentical membrane proteins. Ost4p, the smallest subunit, bridges the catalytic subunit Stt3p with Ost3p. Mutation of transmembrane residues 18-24 in Ost4p has negative effect on OT activity, disrupts the Stt3p-Ost4p-Ost3p complex, results in temperature-sensitive phenotype, and hypoglycosylation. Heterologous expression and purification of integral membrane proteins are the bottleneck in membrane protein research. The authors report the cloning, successful overexpression and purification of recombinant Ost4p with a novel but simple method producing milligram quantities of pure protein. GB1 protein was found to be the most suitable tag for the large scale production of Ost4p. The cleavage of Ost4p conveniently leaves GB1 protein in solution eliminating further purification. The precipitated pure Ost4p is reconstituted in appropriate membrane mimetic. The recombinant protein is highly helical as indicated by the far-UV CD spectrum. The well-dispersed heteronuclear single quantum coherence spectrum indicates that this minimembrane protein is well-folded. The successful production of pure recombinant Ost4p with a novel yet simple method may have important ramification for the production of other membrane proteins.     

Expressing and purifying membrane transport proteins in high yield

Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic.     

Characterization of the Drosophila Atlastin Interactome Reveals VCP as a Functionally Related Interactor

At least 25 genes, many involved in trafficking, localisation or shaping of membrane organelles, have been identified as causative genes for the neurodegenerative disorder hereditary spastic paraplegia (HSP). One of the most commonly mutated HSP genes, atlastin-1, encodes a dynamin-like GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes. However, the molecular mechanisms of atlastin-1-related membrane fusion and axonopathy remain unclear. To better understand its mode of action, we used affinity purification coupled with mass spectrometry to identify protein interactors of atlastin in Drosophila. Analysis of 72 identified proteins revealed that the atlastin interactome contains many proteins involved in protein processing and transport, in addition to proteins with roles in mRNA binding, metabolism and mitochondrial proteins. The highest confidence interactor from mass spectrometry analysis, the ubiquitin-selective AAA-ATPase valosin-containing protein (VCP), was validated as an atlastin-interacting protein, and VCP and atlastin showed overlapping subcellular distributions. Furthermore, VCP acted as a genetic modifier of atlastin: loss of VCP partially suppressed an eye phenotype caused by atlastin overexpression, whereas overexpression of VCP enhanced this phenotype. These interactions between atlastin and VCP suggest a functional relationship between these two proteins, and point to potential shared mechanisms between HSP and other forms of neurodegeneration.