一种利用荧光蛋白mNeonGreen2鉴定EI24蛋白拓扑结构的方法（英文）

方便且精准地检测跨膜蛋白拓扑结构,尤其是跨膜片段的氨基(N-)和羧基(C-)端的朝向,有利于发现新的蛋白质与蛋白质之间的相互作用,并进一步揭示蛋白质重要的生物学功能.自组装荧光蛋白已被广泛用于观察蛋白质与蛋白质之间的相互作用、标记细胞内源蛋白质并实现mRNA定位的可视化.本文扩展了自组装荧光蛋白的应用,将自组装荧光蛋白mNeonGreen2与定点标记技术相结合,以确定跨膜蛋白的拓扑结构.通过该方法,第一次清楚地证明了EI24的N端和C端均朝向细胞质方向.此外,该方法可用于确定定位于其他细胞器且结构尚未解析的跨膜蛋白的拓扑结构.     

应用双分子荧光互补技术分析米曲霉Fus3与Ste12之间的蛋白互作

【背景】双分子荧光互补(Bimolecularfluorescencecomplementation,BiFC)在水稻恶苗病菌(Fusarium fujikuroi)等微生物蛋白互作中的应用已有报道,但在工业菌株米曲霉(Aspergillus oryzae)中还未见应用。【目的】探究米曲霉中Fus3和Ste12蛋白在生长发育中可能存在的相互作用关系,建立在米曲霉活细胞中检测蛋白互作的方法,即BiFC体系。该系统可用于特异性、可视化米曲霉目标蛋白在活细胞中的定位,并且可以更加直观地探究蛋白之间是否存在相互作用。【方法】利用MultisiteGateway复杂载体构建技术,使用切开的绿色荧光蛋白,将荧光蛋白分子的两个片段N端和C端分别与米曲霉Fus3和Ste12蛋白融合,对获得的转化株进行荧光观察。通过BiFC系统检测蛋白之间的相互作用。【结果】成功转化的米曲霉菌丝中观察到荧光,Fus3和Ste12在米曲霉中存在相互作用。【结论】通过BiFC技术证实蛋白质Fus3和Ste12在无性繁殖菌株米曲霉体内发生互作,暗示它们通过互作可能参与除了有性生殖之外的其他细胞功能,并为米曲霉蛋白互作功能研究提供一种新的检测技术和方法。     

双分子荧光互补技术及其在蛋白质相互作用研究中的应用

双分子荧光互补(bimolecularfluorescencecomplementation,BiFC)分析技术,是由Hu等在2002年最先报道的一种直观、快速地判断目标蛋白在活细胞中的定位和相互作用的新技术.该技术巧妙地将荧光蛋白分子的两个互补片段分别与目标蛋白融合表达,如果荧光蛋白活性恢复则表明两目标蛋白发生了相互作用.其后发展出的多色荧光互补技术(multicolorBiFC),不仅能同时检测到多种蛋白质复合体的形成,还能够对不同蛋白质间产生相互作用的强弱进行比较.目前,该技术已用于转录因子,G蛋白βγ亚基的二聚体形式,不同蛋白质间产生相互作用强弱的比较以及蛋白质泛素化等方面的研究工作上.     

基于序列拓扑和二阶隐马尔可夫模型的跨膜蛋白亚细胞定位预测

现有蛋白质亚细胞定位方法针对水溶性蛋白质而设计,对跨膜蛋白并不适用。而专门的跨膜拓扑预测器,又不是为亚细胞定位而设计的。文章改进了跨膜拓扑预测器TMPHMMLoc的模型结构,设计了一个新的二阶隐马尔可夫模型;采用推广到二阶模型的Baum-Welch算法估计模型参数,并把将各个亚细胞位置建立的模型整合为一个预测器。数据集上测试结果表明,此方法性能显著优于针对可溶性蛋白设计的支持向量机方法和模糊k最邻近方法,也优于TMPHMMLoc中提出的隐马尔可夫模型方法,是一个有效的跨膜蛋白亚细胞定位预测方法。     

双分子荧光互补技术

双分子荧光互补（bimolecular fluorescence complementation, BiFC）是近年发展起来的用于体内或体外检测蛋白质相互作用的一项新技术.该技术是将荧光蛋白在合适的位点切开形成不发荧光的2个片段,这2个片段借助融合于其上的目标蛋白的相互作用,彼此靠近,重新形成能具有活性的荧光蛋白.BiFC方法简单直观,既可以检测蛋白之间的相互作用,也可以定位相互作用蛋白质的位点.多色BiFC系统共用或与荧光共振能量转移（FRET）技术联用,还可以检测细胞内多个蛋白质的相互作用.     

利用昆虫杆状病毒表达SARS冠状病毒的刺突蛋白和膜蛋白

SARS冠状病毒是人的严重急性呼吸综合征的病原体。对其他种类冠状病毒的研究结果显示,刺突蛋白(S蛋白)和膜蛋白(M蛋白)是病毒主要的结构蛋白。重组M蛋白和S蛋白可被用来作为抗原检测冠状病毒的感染和制备疫苗。这两个蛋白质分别被克隆并重组到昆虫杆状病毒基因组中,利用重组杆状病毒感染昆虫细胞来表达重组M蛋白和S蛋白,并对M蛋白进行了细胞内定位,融合蛋白的绿色荧光暗示了该蛋白质定位在细胞膜上。     

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。     

超电荷绿色荧光蛋白(+36GFP):一种新型生物大分子穿膜载体

超电荷绿色荧光蛋白(sc GFP)是一种表面带很高净电荷的新型功能蛋白质。ScGFP具有很强的水溶性和抗蛋白质聚集的能力,在生物技术、医药和材料科学方面有着广泛的应用前景。带正电荷的sc GFP能够穿透细胞膜,具有运载核酸和蛋白质等生物大分子进入哺乳动物细胞内的能力。与传统的转运载体相比,sc GFP有细胞毒性低、转运效率高和具广泛的细胞普适性等优点。带正电荷的sc GFP与带负电荷的核酸分子之间通过静电相互作用形成自组装的多离子复合物,这与生物体中的组蛋白和带负电荷的DNA之间自组装成染色质的行为非常相似。本文以带有36个正电荷的超电荷绿色荧光蛋白(+36GFP)为例,对超电荷蛋白的性质,细胞穿透能力以及其作为生物大分子穿透载体的应用等方面做一综述。     

间隙连接蛋白43羧基端在恶性肿瘤中的作用及机制

间隙连接蛋白43(connexin 43,CX43)是间隙连接蛋白家族的重要成员之一,参与体内众多生理和病理过程的调控。结构上,该蛋白由氨基端、跨膜结构及羧基端三部分组成,其羧基端上存在大量蛋白结合位点。通过这些位点,CX43能够与不同的蛋白发生相互作用:一方面,影响CX43自身的磷酸化状态,从而调控其降解、亚细胞定位以及装配等过程;另一方面,CX43羧基端还能够通过某些特定的结合位点,调控其他蛋白分子的功能状态,从而影响信号转导,调节细胞的生物学功能。近年来研究发现,该蛋白的羧基端(carboxyl terminal)显著地影响肿瘤细胞/肿瘤干细胞的生物学特性。该文就CX43羧基端的结构特点、与蛋白质的相互作用位点、调控肿瘤细胞/肿瘤干细胞增殖、迁移、自我更新和成瘤能力的作用机制进行简要综述。     

绿色荧光蛋白的神奇功能

高通量遗传筛选已经揭示出许多潜在的蛋白质·蛋白质之间的相置作用．但这些作用需要进一步验证和研究。在本文中．研究者为解决这一问题．研发出一种简单的分析方法．它整合了含有绿色荧光蛋白（GFP）荧光影像的酵母双杂交系统的威力．可以直接显示活细胞中蛋白质－蛋白质相互作用。这种方法就是荧光双杂交（F2H）分析．它采用了经过修改的lac阻抑物系统,将一种荧光诱饵蛋白固着在染色体lac操纵子阵列．形成一个亮色斑点．具有不同荧光的诱捕融合蛋白质可根据颜色的变化进行共定位分析。     

Assessment of the integral membrane protein topology in living cells

The bimolecular fluorescence complementation (BiFC) phenomenon has been successfully applied for in vivo protein-protein interaction studies and protein tagging analysis. Here we report a novel BiFC-based technique for investigation of integral membrane protein topology in living plant cells. This technique relies on the formation of a fluorescent complex between a non-fluorescent fragment of the yellow fluorescent protein (YFP) targeted into a specific cellular compartment and a counterpart fragment attached to the integral membrane protein N- or C-terminus or inserted into the internal loop(s). We employed this technique for topological studies of beet yellows virus-encoded p6 membrane-embedded movement protein, a protein with known topology, and the potato mop-top virus-encoded integral membrane TGBp2 protein with predicted topology. The results confirm that p6 is a type III integral transmembrane protein. Using a novel method, the central hydrophilic region of TGBp2 was localized into the ER lumen, whereas the N- and C-termini localized to the cytosol. We conclude that the BiFC-based reporter system for membrane protein topology analysis is a relatively fast and efficient method that can be used for high-throughput analysis of proteins integrated into the endoplasmic reticulum in living plant cells.     

Split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts

We developed a split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts. In this assay, the N- and C-terminal fragments of Renilla reniforms luciferase are translationally fused to bait and prey proteins, respectively. When the proteins interact, split luciferase becomes activated and emits luminescence that can be measured by a microplate luminometer. Split luciferase activity was measured by first transforming protoplasts with a DNA vector in a 96-well plate. DNA vector expressing both bait and prey genes was constructed through two independent in vitro DNA recombinant reactions, Gateway and Cre-loxP. As proof of concept, we detected the protein-protein interactions between the nuclear histones 2A and 2B, as well as between membrane proteins SYP (syntaxin of plant) 51 and SYP61, in Arabidopsis protoplasts.     

The GxxxG-containing transmembrane domain of the CCK4 oncogene does not encode preferential self-interactions

The recently cloned colon carcinoma kinase 4 (CCK4) oncogene contains an evolutionarily conserved GxxxG motif in its single transmembrane domain (TMD). It has previously been suggested that this pairwise glycine motif may provide a strong driving force for transmembrane helix-helix interactions. Since CCK4 is thought to represent a new member of the receptor tyrosine kinase family, interactions between the TMDs may be important in receptor self-association and activation of signal transduction pathways. To determine whether this conserved CCK4 TMD can drive protein-protein interactions, we have carried out a thermodynamic study using the TMD expressed as a Staphylococcal nuclease (SN) fusion protein. Similar SN-TMD fusion proteins have been used to determine the sequence specificity and thermodynamics of transmembrane helix-helix interactions in a number of membrane proteins, including glycophorin A. Using sedimentation equilibrium in C14 betaine micelles, we discovered that the CCK4 TMD is unable to drive strong protein-protein interactions. At high protein/detergent ratios, the SN-CCK4 fusion protein will dimerize, but a stochastic model for protein association in micelles can explain the observed dimer population. For low-affinity interactions such as the one studied here, an understanding of this discrete stochastic distribution of membrane proteins in micelles is important for distinguishing between preferential and random self-interactions, which can both influence the oligomeric population. The lack of a thermodynamically meaningful self-association propensity for the CCK4 TMDs demonstrates that a GxxxG motif is not sufficient to drive transmembrane helix-helix interactions.     

N-terminal tail export from the mitochondrial matrix. Adherence to the prokaryotic "positive-inside" rule of membrane protein topology.

Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N- and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for sec-independent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery.     

Non-invasive topology analysis of membrane proteins in the secretory pathway

We present a novel method to experimentally visualize in vivo the topology of transmembrane proteins residing in the endoplasmic reticulum (ER) membrane or passing through the secretory pathway on their way to their final destination. This approach, so-called redox-based topology analysis (ReTA), is based on fusion of transmembrane proteins with redox-sensitive GFP (roGFP) and ratiometric imaging. The ratio images provide direct information on the orientation of roGFP relative to the membrane as the roGFP fluorescence alters with changes in the glutathione redox potential across the ER membrane. As proof of concept, we produced binary read-outs using oxidized roGFP inside the ER lumen and reduced roGFP on the cytosolic side of the membrane for both N- and C-terminal fusions of single and multi-spanning membrane proteins. Further, successive deletion of hydrophobic domains from the C-terminus of the K/HDEL receptor ERD2 resulted in alternating localization of roGFP and a topology model for At ERD2 with six transmembrane domains.     

Transmembrane Nox4 topology revealed by topological determination by Ubiquitin Fusion Assay,a novel method to uncover membrane protein topology

The NADPH oxidase Nox4 is a multi-pass membrane protein responsible for the generation of reactive oxygen species that are implicated in cellular signaling but may also cause pathological situations when dysregulated. Although topological organization of integral membrane protein dictates its function, only limited experimental data describing Nox4's topology are available.To provide deeper insight on Nox4 structural organization, we developed a novel method to determinate membrane protein topology in their cellular environment, named Topological Determination by Ubiquitin Fusion Assay (ToDUFA). It is based on the proteolytic capacity of the deubiquitinase enzymes to process ubiquitin fusion proteins. This straightforward method, validated on two well-known protein's topologies (IL1RI and Nox2), allowed us to discriminate rapidly the topological orientation of protein's domains facing either the nucleocytosolic or the exterior/luminal compartments. Using this method, we were able for the first time to determine experimentally the topology of Nox4 which consists of 6 transmembrane domains with its N- and C-terminus moieties facing the cytosol. While the first, third and fifth loops of Nox4 protein are extracellular; the second and fourth loops are located in the cytosolic side. This approach can be easily extended to characterize the topology of all others members of the NADPH oxidase family or any multi-pass membrane proteins.Considering the importance of protein topology knowledge in cell biology research and pharmacological development, we believe that this novel method will represent a widely useful technique to easily uncover complex membrane protein's topology.     

Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli

A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structure-function relationships. Although these maps can be predicted directly from amino acid sequence, the predictions are more accurate if combined with experimental data, which are usually obtained by fusing a reporter protein to the C-terminus of the protein. However, as reporter proteins are large, they cannot be used to report on the cytoplasmic/periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli.     

Transmembrane topology of PiT-2, a phosphate transporter-retrovirus receptor

PiT-1 and PiT-2 are related multiple transmembrane proteins which function as sodium-dependent phosphate transporters and as the cell receptors of several oncoretroviruses. Two copies of a homology domain that is found in distantly related species assign these proteins to a large family of phosphate transporters. A current membrane topology model of PiT-1 and PiT-2 predicts 10 transmembrane domains. However, the validity of this model has not been addressed experimentally. We addressed this issue by a comprehensive study of human PiT-2. Evidence was obtained for glycosylation of asparagine 81. Epitope tagging showed that the N- and C-terminal extremities are extracellular. The orientation of C-terminal-truncation mutants expressed in cell-free translation assays and incorporated into microsomal membranes was examined by immunoprecipitation. Data were interpreted with respect to previous knowledge about retrovirus binding sites, to the existence of repeated homology domains, and to predictions made in family members. A model in which PiT-2 has 12 transmembrane domains and extracellular N- and C-terminal extremities is proposed. This model, which differs significantly from previous predictions about PiT-2 topology, may be useful for further investigations of PiT-2 interactions with other proteins and for the understanding of PiT-2 transporter and virus receptor functions.     

Molecular simulations of lipid-mediated protein-protein interactions

Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved.     

Sequence similarity as a predictor of the transmembrane topology of membrane-intrinsic subunits of bacterial respiratory chain enzymes

Integral membrane proteins usually have a predominantly alpha-helical secondary structure in which transmembrane segments are connected by membrane-extrinsic loops. Although a number of membrane protein structures have been reported in recent years, in most cases transmembrane topologies are initially predicted using a variety of theoretical techniques, including hydropathy analyses and the "positive inside" rule. We have explored the use of plots of the distribution of sequence similarity within families of membrane proteins comprising homeomorphic domains as a new method for the prediction/verification of the orientation of transmembrane topology models within certain families of multimeric respiratory chain enzymes. Within such proteins, analyses of sequence similarity can: i) identify heme and/or quinol binding sites; ii) identify potential electron-transfer conduits to/from prosthetic groups; and iii) locate regions defining potential subunit-subunit interactions. We mined emerging bioinformatic data for sequences of 11 families of membrane-intrinsic proteins that are part of multimeric respiratory chain complexes that also have membrane-extrinsic subunits. The sequences of each family were then aligned and the resultant alignments converted into a graphical format recording an empirical measure of the sequence similarity plotted versus residue position. In each case, this plot was compared to the predicted transmembrane topology. With one exception, there is a strong correlation between the existence