Prediction of Protein Binding Regions in Disordered Proteins

Many disordered proteins function via binding to a structured partner and undergo a disorder-to-order transition. The coupled folding and binding can confer several functional advantages such as the precise control of binding specificity without increased affinity. Additionally, the inherent flexibility allows the binding site to adopt various conformations and to bind to multiple partners. These features explain the prevalence of such binding elements in signaling and regulatory processes. In this work, we report ANCHOR, a method for the prediction of disordered binding regions. ANCHOR relies on the pairwise energy estimation approach that is the basis of IUPred, a previous general disorder prediction method. In order to predict disordered binding regions, we seek to identify segments that are in disordered regions, cannot form enough favorable intrachain interactions to fold on their own, and are likely to gain stabilizing energy by interacting with a globular protein partner. The performance of ANCHOR was found to be largely independent from the amino acid composition and adopted secondary structure. Longer binding sites generally were predicted to be segmented, in agreement with available experimentally characterized examples. Scanning several hundred proteomes showed that the occurrence of disordered binding sites increased with the complexity of the organisms even compared to disordered regions in general. Furthermore, the length distribution of binding sites was different from disordered protein regions in general and was dominated by shorter segments. These results underline the importance of disordered proteins and protein segments in establishing new binding regions. Due to their specific biophysical properties, disordered binding sites generally carry a robust sequence signal, and this signal is efficiently captured by our method. Through its generality, ANCHOR opens new ways to study the essential functional sites of disordered proteins.     

Topological Comparison of Two Helix Destabilizing Proteins: Ribonuclease A and the Gene 5 DNA Binding Protein

Abstract

A topological comparison of the two helix destabilizing proteins, pancreatic ribonuclease A and the gene S DNA binding protein of bacteriophage fd has been completed utilizing the available high resolution tertiary structures of each protein. The results indicate these two proteins are structurally if not also evolutionarily related. Regions of closest topological equivalence occur between beta loops directly involved in nucleotide binding or are required for the maintenance of their respective oligonucleotide binding channels. In addition, there is a similar placement of critical amino acid side chains about the binding site. Further evidence for this structural relationship is obtained by comparison of structural data for the mode of complexation of polynucleotides to each protein. The results of topological comparison suggest the essential property shared by helix destabilizing proteins, whether specialized DNA binding proteins such as G5BP or proteins with other primary functional roles, like ribonuclease A, is the presence of an elongated oligonucleotide binding channel. Although ribonuclease A and G5BP are structurally related, it seems likely any protein with this structural feature will exhibit a helix destabilizing capacity. This conclusion is supported by the diversity of molecular characteristics shown by other proteins having this activity.     

转录因子X-box结合蛋白1相互作用蛋白的筛选和鉴定

X-box 结合蛋白 1 是一种重要的转录因子,参与体内多项信号转导过程. 为进一步研究 XBP1 的生物学功能,运用酵母双杂交技术在肝细胞文库中筛选 XBP1 的结合蛋白. 首先运用 PCR 技术扩增获得 XBP1 的编码序列,克隆至 pGEM-T 载体,经测序鉴定后,亚克隆至诱饵载体 pGBKT7 中,转化酵母 AH109(a type). 免疫印迹检测诱饵质粒 pGBKT7-XBP1 在AH109 酵母中的表达之后,含有诱饵质粒的酵母 AH109 与含有肝细胞 cDNA 文库质粒 pACT2 的酵母 Y187(αtype)配合,配合后的二倍体酵母生长在含有 X-α-gal 的营养缺陷型培养基上 (SD/-Trp-Leu-His-Ade) 进行选择和筛选,经测序和序列比对确定阳性克隆的开放读码框 ORF,得到 7 种不同的蛋白质. 为了进一步验证这些筛选蛋白质与 XBP1 的相互作用,克隆其中一种蛋白质 MT1E,并运用 GST pulldown 和免疫共沉淀技术成功检测了 MT1E 和 XBP1 的相互作用(体外 / 体内),结果提示,MT1E 可能是 XBP1 的一个新的调节蛋白. 通过酵母双杂交技术筛选得到的 7 种蛋白质分别与肝细胞基础代谢、蛋白质的合成与运输、细胞的增殖与凋亡密切相关. 上述结果有助于揭示 XBP1 的生物学功能,为进一步探讨 XBP1 的表达和调控机制提供新线索.     

Characterization of the Crithidia fasciculata mRNA Cycling Sequence Binding Proteins

The Crithidia fasciculata cycling sequence binding protein (CSBP) binds with high specificity to sequence elements in several mRNAs that accumulate periodically during the cell cycle. Mutations in these sequence elements abolish both cycling of the mRNA and binding of CSBP. Two genes, CSBPA and CSBPB, encoding putative subunits of CSBP have been cloned and were found to be present in tandem on the same DNA molecule and to be closely related. CSBPA and CSBPB are predicted to encode proteins with sizes of 35.6 and 42.0 kDa, respectively. Both CSBPA and CSBPB proteins have a predicted coiled-coil domain near the N terminus and a novel histidine and cysteine motif near the C terminus. The latter motif is conserved in other trypanosomatid species. Gel sieving chromatography and glycerol gradient sedimentation results indicate that CSBP has a molecular mass in excess of 200 kDa and an extended structure. Recombinant CSBPA and CSBPB also bind specifically to the cycling sequence and together can be reconstituted to give an RNA gel shift similar to that of purified CSBP. Proteins in cell extracts bind to an RNA probe containing six copies of the cycling sequence. The RNA-protein complexes contain both CSBPA and CSBPB, and the binding activity cycles in near synchrony with target mRNA levels. CSBPA and CSBPB mRNA and protein levels show little variation throughout the cell cycle, suggesting that additional factors are involved in the cyclic binding to the cycling sequence elements.     

光亲和标记鉴定玉米根脱落酸结合蛋白

光亲和标记鉴定玉米根脱落酸结合蛋白吴忠义,陈珈,朱美君（北京农业大学生物学院,１０００９４）关键词结合蛋白;光亲和标记;ＡＢＡ;受体;微粒体脱落酸（ＡＢＡ）作为一大类植物激素,在高等植物的生长发育以及对逆境的适应过程中发挥着重要作用。在探讨激素作用的...     

神经元蛋白3.1结合蛋白的筛选与鉴定

哺乳动物肺组织发育的基本模式总体分为上皮分支的形态发生和分隔膜的形成两个部分．基因p311是克隆到的一个在分隔膜形成阶段特异表达的基因,可能在肺泡发育中起重要作用．为进一步探讨神经元蛋白3.1(P311)对肺发育过程的影响,构建了小鼠肺组织 cDNA文库,以融合Gal4 DNA结合结构域的重组P311蛋白为诱饵,利用酵母双杂交技术从文库中筛选P311结合蛋白．通过免疫共沉淀和双分子荧光互补等技术进一步验证, SPARC(secreted protein, acidic and rich in cysteine)被确定为P311 相互作用蛋白．进一步的研究发现,SPARC在肺组织中具有与P311相似的表达时序特征,双重免疫组织化学染色显示SPARC和P311在小鼠肺组织中共定位于肺泡上皮细胞和肌成纤维细胞中．提示P311可能通过与SPARC的相互作用影响肺泡发育．     

Small Abundant DNA Binding Proteins from the Thermoacidophilic Archaeon Sulfolobus shibatae Constrain Negative DNA Supercoils

Major DNA binding proteins, designated Ssh7, were purified from the thermoacidophilic archaeon Sulfolobus shibatae. The Ssh7 proteins have an apparent molecular mass of 6.5 kDa and are similar to the 7-kDa DNA binding proteins from Sulfolobus acidocaldarius and Sulfolobus solfataricus in N-terminal amino acid sequence. The proteins constitute about 4.8% of the cellular protein. Upon binding to DNA, the Ssh7 proteins constrain negative supercoils. At the tested Ssh7/DNA mass ratios (0 to 1.65), one negative supercoil was taken up by approximately 20 Ssh7 molecules. Our results, together with the observation that the viral DNA isolated from S. shibatae is relaxed, suggest that regions of free DNA in the S. shibatae genome, if present, are highly positively supercoiled.     

Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.     

Conformational Properties of the TATA-Box Binding Sequence of DNA

Abstract

Nanosecond scale molecular dynamics simulations in water demonstrate that the DNA oligomer, GCGTATATAAAACGC, which contains a target site for the TATA-box binding protein (TBP), has an intrinsic preference to adopt an A-like conformation in the region of the TATA-box and undergoes bending related to that seen within in the TBP complex. This result is obtained from two independent simulations using different starting structures. In line with earlier suggestions of Guzikevich-Guerstein and Shakked, these simulations imply that an A-DNA conformation may be an important intermediate step in forming the strongly distorted DNA observed within the crystallographically determined complex with TBP. These results also support modeling studies by Lebrun et al. which suggest that the TBP binding mechanism can be broken down into a backbone transition to an A-like form coupled with a mechanical distortion which locally stretches and unwinds the DNA.     

Novel DNA Binding Properties of the Mcm10 Protein from Saccharomyces cerevisiae

The Mcm10 protein is essential for chromosomal DNA replication in eukaryotic cells. We purified the Saccharomyces cerevisiae Mcm10 (ScMcm10) and characterized its DNA binding properties. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that ScMcm10 binds stably to both double strand (ds) DNA and single strand (ss) DNA. On short DNA templates of 25 or 50 bp, surface plasmon resonance analysis showed a ∼1:1 stoichiometry of ScMcm10 to dsDNA. On longer dsDNA templates, however, multiple copies of ScMcm10 cooperated in the rapid assembly of a large, stable nucleoprotein complex. The amount of protein bound was directly proportional to the length of the DNA, with an average occupancy spacing of 21–24 bp. This tight spacing is consistent with a nucleoprotein structure in which ScMcm10 is aligned along the helical axis of the dsDNA. In contrast, the stoichiometry of ScMcm10 bound to ssDNA of 20–50 nucleotides was ∼3:1 suggesting that interaction with ssDNA induces the assembly of a multisubunit ScMcm10 complex composed of at least three subunits. The tight packing of ScMcm10 on dsDNA and the assembly of a multisubunit complex on ssDNA suggests that, in addition to protein-DNA, protein-protein interactions may be involved in forming the nucleoprotein complex. We propose that these DNA binding properties have an important role in (i) initiation of DNA replication and (ii) formation and maintenance of a stable replication fork during the elongation phase of chromosomal DNA replication.MCM10 is a ubiquitous, conserved gene essential for DNA replication in eukaryotes. It was first discovered in yeast genetic screens designed to detect mutants defective in DNA synthesis and minichromosome maintenance (1, 2). In vivo, Mcm10 associates with chromatin and chromosomal replication origins in human cells (hMcm10), Xenopus laevis (XMcm10), Schizosaccharomyces pombe (SpMcm10), and Saccharomyces cerevisiae (ScMcm10) (3–6). In S. cerevisiae, initiation of chromosomal replication occurs at multiple specific sites known as autonomously replicating sequences (ARSs)² (7). Mutations in MCM10 enhance the loss rate of plasmids bearing specific ARSs (8), suggesting a function for ScMcm10 in initiation.In eukaryotic systems replication initiation is a cell cycle-regulated process characterized by a multistep sequential loading of ORC, Cdc6, Cdt1, and the Mcm2–7 complex onto the origin in G₁ to form the pre-RC complex. Binding of ORC, Cdc6p, and Cdt1p to chromatin is a prerequisite for the recruitment of Mcm2–7 (9, 10). The next step in the assembly of the initiation replication apparatus in S. cerevisiae involves protein kinases (Cdc28 and Cdc7/Dbf4), and recruitment of Mcm10, Cdc45, and the GINS complex. The mechanism for targeting Mcm10 to replications origins is unknown. However, recent studies in S. cerevisiae have shown that Mcm10 and Mcm2–7 physically interact (6, 11). It is now believed that in late G₁, chromatin-bound Mcm2–7 is responsible for the recruitment of Mcm10 presumably via protein-protein interactions (12). Prior studies in the Xenopus laevis system reached similar conclusions (4). Additional interactions of Mcm10 with other components of the pre-RC cannot be excluded (13).A key step in the initiation of replication is the local melting of an origin DNA sequence, which occurs at the G₁/S transition and throughout the S phase. The mechanism of this essential DNA-melting process is not understood. There is no evidence in S. cerevisiae that the assembled pre-RC complex leads to the melting of an origin DNA sequence. This unwinding may occur only following the recruitment of Mcm10, raising the possibility that Mcm10 is a key component of the initiation machinery responsible for this process. Results of a study in the Xenopus egg extract system (4), which showed that omission of XMcm10 blocks unwinding of plasmid DNA and initiation of DNA replication, are consistent with this notion. An additional function of Mcm10 in initiation is in the recruitment of Cdc45 to replication origins, presumably via Mcm10-Cdc45 physical interactions (5, 14). Cdc45 is believed to be important for the activation of replication origins and the assembly of the replication elongation complex (15). Upon initiation of DNA replication, ScMcm10 moves from the origin to origin-proximal sequences suggesting that ScMcm10 associates with moving replication forks (12) and is consistent with the observation that elevated temperatures cause pausing of replication forks in a mcm10-1 ts mutant (8). Both ScMcm10 and SpMcm10 interact with DNA polymerase α supporting the notion that replication fork movement requires Mcm10. ScMcm10 and polymerase α form a complex on and off the DNA in vivo (12). In S. pombe, SpMcm10 stimulates the activity of polymerase α in vitro and associates with a primase activity (16, 17) that has not been reported in other eukaryotes (18).Previous studies with Mcm10 in other systems showed that Mcm10 binds DNA. Using a filter binding assay Fien and Hurwitz (16) reported that SpMcm10 from S. pombe binds well to ssDNA but barely interacts (20-fold lower affinity) with dsDNA. It has been suggested that binding of SpMcm10 to ssDNA is important for the recruitment of polymerase α (16). Recently, it has been reported that a DNA binding activity is also associated with XMcm10 protein from X. laevis. Measurements of fluorescence anisotropy were used to show binding of XMcm10 to short, 25-nucleotide-long oligonucleotides (18). These studies have shown that XMcm10 has similar affinities for ssDNA and dsDNA. Unlike SpMcm10, which harbors a single DNA-binding domain in the N-terminal half of the protein, XMcm10 seems to contain two distinct domains for binding DNA. The biological implication of having two DNA-binding domains is not clear.It appears that there are differences in the quaternary structure of Mcm10 from different organisms. Although SpMcm10 and XMcm10 may be a homodimer in solution (17, 18), a recent electron microscopy study suggested that human hMcm10 has a hexameric ring structure (19). The same study reported that hMcm10 interacts with ssDNA but failed to bind dsDNA. The differences in structure and DNA binding properties may reflect differences in the function of Mcm10 in various organisms as well as in the protein preparations.Here we report, for the first time, the characterization of the DNA binding properties of purified Mcm10 from S. cerevisiae. We show that ScMcm10 forms a stable complex with dsDNA and ssDNA. In addition, we demonstrate that dsDNA longer than 50 bp sustains oligomerization of ScMcm10. The number of ScMcm10 molecules bound is directly proportional to the size of the dsDNA, suggesting that ScMcm10 is tightly packed on the dsDNA, perhaps in a head to tail oligomeric structure. In contrast to a 25-bp-long dsDNA, which supports the binding of a single copy of ScMcm10, ssDNA containing only 20 nucleotides may sustain binding of as many as three copies of ScMcm10, suggesting that a ScMcm10 complex composed of at least 3 subunits assembles on ssDNA. We believe that these distinct binding properties to dsDNA and ssDNA are important for the ScMcm10 functions in initiation, formation of replication forks, and the maintenance of replication fork progression during chromosomal DNA replication.     

膜上tRNA结合蛋白的分离与初步鉴定

用ＴｒｉｔｏｎＸ－１１４分相法分离啤酒酵母的膜总蛋白,经过酵母ｔＲＮＡ分子交联的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,用０－０．８ｍｏｌ／Ｌ（ＮＨ４０２ＳＯ４梯度缓冲液洗脱ｔＲＮＡ结合的蛋白质。凝胶阻滞电泳实验室鉴定出两种主要的与ｔＲＮＡ分子特异性结合的蛋白质。     

用于筛选人工结合蛋白的骨架蛋白

抗体作为最著名的天然结合蛋白,因其具有与抗原特异结合的特性,近100多年以来无论在生物技术领域,还是在疾病的诊断及治疗方面,都发挥着广泛而重要的作用。但是抗体自身固有的局限性也在很大程度上限制了它的应用,而人工结合蛋白既具有抗体的特点,又兼具更多优势:分子更小;稳定性更高;能在大肠杆菌中高产量、高可溶性表达;易于修饰;能够达到高亲合力和高特异性;并且与抗体没有知识产权的冲突,因此被称为理想的"新一代抗体"。人工结合蛋白是从基因改造构建而成的骨架蛋白库中针对特定的靶分子筛选而得的。从骨架蛋白的概念和设计,骨架蛋白的分类,应用骨架蛋白筛选人工结合蛋白的技术以及人工结合蛋白的应用和前景等方面进行总结概述。