结核分枝杆菌Hsp16.3单克隆抗体的制备及其特性鉴定

制备抗结核分枝杆菌Hsp16.3的单克隆抗体,并对其生物学特性进行鉴定。将含目的基因的表达载体pProEXHTb-Hsp16.3,通过E.coli DH5α诱导表达,获得含有6譎is的Hsp16.3蛋白,采用Ni-NTA纯化试剂盒进行目的蛋白的纯化,并用透析方法进行蛋白复性。将复性的蛋白免疫BALB/c小鼠,利用杂交瘤技术进行细胞融合,间接ELISA筛选阳性杂交瘤细胞株。将获得的Hsp16.3阳性杂交瘤细胞株分别利用间接ELISA法和Western blot方法进行效价、相对亲和力及特异性的测定。获得了三株Hsp16.3的单克隆抗体,分别命名为3H6F9、1D5E1和4H8G6,其效价分别为1:1?07、1:1?06和1:1?06,相对亲和力分别为0.0001 mg/mL、0.001 mg/mL和0.001 mg/mL,并且无交叉反应性。所获得的结核分枝杆菌Hsp16.3的单克隆抗体效价高、特异性强,为进一步研究Hsp16.3在结核分枝杆菌潜伏感染中的作用提供了有效的工具。     

Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex

Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle.     

Complementation of transforming domains in E1a/myc chimaeras.

The myc oncogene is functionally similar to adenovirus E1a in its ability to collaborate with activated ras oncogenes to transform primary fibroblasts. The transforming functions of E1a and myc have been mapped to two distinct regions in each protein. I investigated the functional similarities between E1a and myc by constructing E1a/myc chimaeras to discover whether the individual transforming domains of E1a could complement individual myc-transforming domains. Transformation assays in rat embryo fibroblasts demonstrated that the N-terminal transforming domain of E1a (CR1) could complement the C-terminal transforming domain of myc in cis, and that the reciprocal chimaera (N-terminal myc/C-terminal E1a) was also active. Chimaeras constructed using domains from transformation-defective mutants of either E1a or myc were inactive, indicating that both E1a and myc domains contribute to function. These experiments suggest that transformation by myc and E1a may involve interactions with common substrates.     

复性的乙型肝炎病毒X蛋白突变体HBxΔNΔC具有体外生物学活性

乙型肝炎病毒X蛋白(HBx)在肝炎引起的肝癌病变中作为一个多功能调节因子起着关键的作用.对HBx的功能有非常多的体外研究报导,而由于HBx蛋白难以溶解和稳定使得体外的研究报导非常少.我们根据肝癌患者中最常见的截短突变体,在大肠杆菌包涵体中重组表达了一个保留了第12～127位氨基酸的、N端和C端截断的突变体HBxΔNΔC,并使用变复性的方法将其复性溶解.分子筛显示HBxΔNΔC为单体;圆二色谱显示HBxΔNΔC包含有5％的α-螺旋,30％的β-折叠和65％的其他结构.复性的HBxΔNΔC能从人HepG2细胞中拉下(pull-down)肿瘤抑制因子p53和DDB1蛋白,它还能刺激肝细胞的迁移.腹腔注射1μg/g的HBxΔNΔC能明显刺激小鼠肝的生长.这些结果显示HBxΔNΔC具有一定的生物学活性,它可以用来研究体外的HBx结构和分子作用机制.     

Convergent evolution of similar function in two structurally divergent enzymes

An example of two related enzymes that catalyse similar reactions but possess different active sites is provided by comparing the structure of Escherichia coli thioredoxin reductase with glutathione reductase. Both are dimeric enzymes that catalyse the reduction of disulphides by pyridine nucleotides through an enzyme disulphide and a flavin. Human glutathione reductase contains four structural domains within each molecule: the flavin-adenine dinucleotide (FAD)- and nicotinamide-adenine dinucleotide phosphate (NADPH)-binding domains, the 'central' domain and the C-terminal domain that provides the dimer interface and part of the active site. Although both enzymes share the same catalytic mechanism and similar tertiary structures, their active sites do not resemble each other. We have determined the crystal structure of E. coli thioredoxin reductase at 2 A resolution, and show that thioredoxin reductase lacks the domain that provides the dimer interface in glutathione reductase, and forms a completely different dimeric structure. The catalytically active disulphides are located in different domains on opposite sides of the flavin ring system. This suggests that these enzymes diverged from an ancestral nucleotide-binding protein and acquired their disulphide reductase activities independently.     

Structure and mechanism of the hexameric MecA-ClpC molecular machine

Regulated proteolysis by ATP-dependent proteases is universal in all living cells. Bacterial ClpC, a member of the Clp/Hsp100 family of AAA+ proteins (ATPases associated with diverse cellular activities) with two nucleotide-binding domains (D1 and D2), requires the adaptor protein MecA for activation and substrate targeting. The activated, hexameric MecA-ClpC molecular machine harnesses the energy of ATP binding and hydrolysis to unfold specific substrate proteins and translocate the unfolded polypeptide to the ClpP protease for degradation. Here we report three related crystal structures: a heterodimer between MecA and the amino domain of ClpC, a heterododecamer between MecA and D2-deleted ClpC, and a hexameric complex between MecA and full-length ClpC. In conjunction with biochemical analyses, these structures reveal the organizational principles behind the hexameric MecA-ClpC complex, explain the molecular mechanisms for MecA-mediated ClpC activation and provide mechanistic insights into the function of the MecA-ClpC molecular machine. These findings have implications for related Clp/Hsp100 molecular machines.     

Structure of the calcium regulatory muscle protein troponin-C at 2.8 A resolution

Crystals of turkey skeletal muscle troponin-C reveal a molecule of two domains with an unusual structure. Two Ca2+ ions are bound to the C-terminal domain. The two cation-binding sites of the regulatory (N-terminal) domain are Ca2+ free; this domain adopts a markedly different conformation from the C-terminal domain. The two domains are connected by a long nine-turn alpha-helix; three of these turns are exposed fully to solvent.     

Hsp90 chaperones protein folding in vitro.

The heat-shock protein Hsp90 is the most abundant constitutively expressed stress protein in the cytosol of eukaryotic cells, where it participates in the maturation of other proteins, modulation of protein activity in the case of hormone-free steroid receptors, and intracellular transport of some newly synthesized kinases. A feature of all these processes could be their dependence on the formation of protein structure. If Hsp90 is a molecular chaperone involved in maintaining a certain subset of cellular proteins in an inactive form, it should also be able to recognize and bind non-native proteins, thereby influencing their folding to the native state. Here we investigate whether Hsp90 can influence protein folding in vitro and show that Hsp90 suppresses the formation of protein aggregates by binding to the target proteins at a stoichiometry of one Hsp90 dimer to one or two substrate molecule(s). Furthermore, the yield of correctly folded and functional protein is increased significantly. The action of Hsp90 does not depend on the presence of nucleoside triphosphates, so it may be that Hsp90 uses a novel molecular mechanism to assist protein folding in vivo.     

Unfolding of C2A Domain of Synaptotagmin I in the Presence of Guanidine Hydrochloride

The C2 domain originally referred to the second of four constant structural motifs in protein kinase C (PKC). Now this domain represents a large structural family sharing a homologous dimensional structure in many proteins that play important roles in many organisms. The C2A domain is one of the two C2 domains of synaptotagmin I involved in the Ca^2 regulation of exocytosis. This domain is mostly composed of β-sheet except for a small fraction of α-helix, and therefore provides an ideal model for a protein folding study. In this report, the unfolding equilibrium of the C2A domain in guanidine hydrochloride (GdnHCI) containing solutions has been studied using ultraviolet (UV) difference spectrum, fluorescence spectrum, size exclusion chromatography (SEC), and circular dichroism (CD) spectrum. The results suggest that unfolding of the C2A domain occurs as a two-state process during GdnHCI titration. By examining the changes of both tertiary structure and secondary structure, no intermediates could be detected during this unfolding study. However, it has been found that the native state of the C2A domain has a large hydrophobic surface. This result suggests that as a fragment of a protein, the C2A domain itself may exist in a state with large hydrophobic surface. This hydrophobic surface may be the molecular basis for interaction between domains in the whole protein.Furthermore, the hydrophobic behavior may play a role during the oligomerization of svnaptotagmin.     

Reductase activity of human thioredoxin-like protein and its effects on cell apoptosis

Thioredoxin is a ubiquitous dithiol oxidoreductase found in many organisms and involved in numerous biochemical processes. Human thioredoxin-like protein (hTRXL) is differentially expressed at different development stages of human fetal cerebrum and belongs to an expanding family of thioredoxins. Recombinant hTRXL and truncated hTRXLs corresponding to the N-terminal (hTRXL-N) and C-terminal (hTRXL-C) domains are expressed and purified. In insulin disulfide reduction assay, both full-length hTRXL and hTRXL-N show reducing activity for the insulin disulfide bonds. As expected, the hTRXL-C failed to reduce insulin. MCF-7 cell stably transfected with hTRXL cDNA exhibits increased sensitivity to apoptosis induced by phorbol myristic acetate (PMA) and ionomysin.     

Structural and functional conservation of key domains in InsP3 and ryanodine receptors

Inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca(2+) channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP(3)R gating is initiated by InsP(3) binding to the InsP(3)-binding core (IBC, residues 224-604 of InsP(3)R1) and it requires the suppressor domain (SD, residues 1-223 of InsP(3)R1). Here we present structures of the amino-terminal region (NT, residues 1-604) of rat InsP(3)R1 with (3.6??) and without (3.0??) InsP(3) bound. The arrangement of the three NT domains, SD, IBC-β and IBC-α, identifies two discrete interfaces (α and β) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP(3)R and of the ABC domains docked into RyR are remarkably similar. The importance of the α-interface for activation of InsP(3)R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP(3) causes partial closure of the clam-like IBC, disrupting the β-interface and pulling the SD towards the IBC. This reorients an exposed SD loop ('hotspot' (HS) loop) that is essential for InsP(3)R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length InsP(3)R, and an InsP(3)R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP(3) and blocked by ryanodine. Activation mechanisms are conserved between InsP(3)R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit reorients the first domain (SD or A domain), allowing it, through interactions of the second domain of an adjacent subunit (IBC-β or B domain), to gate the pore.     

Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase

Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface. The evolutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosphorylated tyrosine residues and has cell-transforming activity. Point mutations in Cbl that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential. In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-70. Here we describe the crystal structure of Cbl-N, both alone and in complex with a phosphopeptide that represents its binding site in ZAP-70. The structures show that Cbl-N is composed of three interacting domains: a four-helix bundle (4H), an EF-hand calcium-binding domain, and a divergent SH2 domain that was not recognizable from the amino-acid sequence of the protein. The calcium-bound EF hand wedges between the 4H and SH2 domains and roughly determines their relative orientation. In the ligand-occupied structure, the 4H domain packs against the SH2 domain and completes its phosphotyrosine-recognition pocket. Disruption of this binding to ZAP-70 as a result of structure-based mutations in the 4H, EF-hand and SH2 domains confirms that the three domains together form an integrated phosphoprotein-recognition module.     

Effects of truncated mutants of the ε subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability

The ε subunit of the chloroplast ATP synthase and the truncated ε mutants which lack some amino acid residues from the N-terminus or C-terminus were overexpressed in E. coil When the ε subunit or the truncated ε proteins was added to the spinach chloroplast suspension, both the intensity of the fast phase of millisecond delayed light emission (ms-DLE) and the cyclic and noncyclic photophosphorylation activity of chloroplast were enhanced. With an increase in the number of residues deleted from the N-terminus, the enhancement effect of the N-terminal truncated proteins decreased gradually. For the C-terminal truncated proteins, the enhancement effect increased gradually with an increase in the number of residues deleted from the C-terminus. Besides, the ATP synthesis activity of ε-deficient membrane reconstituted with the ε subunit or the truncated ε proteins was compared. The ATP synthesis activity of reconstituted membrane with the N-terminal truncated proteins decreased gradually as the number of residues deleted from the N-terminus increased. For the C-terminal truncated proteins, the ATP synthesis activity of reconstituted membrane increased gradually with an increase in the number of residues deleted from the C-terminus, but was still lower than that of the wild type ε protein. These results suggested that: (a) the N-terminal domain of the ε subunit of the chloroplast ATP synthase could affect the ATP synthesis activity of ATP synthase by regulating the efficiency of blocking proton leakage of ε subunit; and (b) the C-terminal domain of the ε subunit of the chloroplast ATP synthase had a subtle function in modulating the ATP synthesis ability of ATP synthase.     

Insulin gene enhancer binding protein Isl-1 is a member of a novel class of proteins containing both a homeo- and a Cys-His domain.

The activity of the rat insulin I gene enhancer is mainly dependent on two cis-acting protein-binding domains. Here we report the isolation of a complementary DNA encoding a protein, Isl-1, that binds to one of these domains. Isl-1 contains a homeodomain with greatest similarity to those of the Caenorhabditis elegans proteins encoded by mec-3 and lin-11. In addition, Isl-1, like the lin-11 and mec-3 gene products, contains a novel Cys-His domain which is reminiscent of known metal-binding regions. Together these proteins define a novel class of proteins containing both a homeo- and a Cys His-domain. Isl-1 is preferentially expressed in cells of pancreatic endocrine origin. If the structural homologies between Isl-1 and the C. elegans gene products reflect functional similarities, a role for Isl-1 in the development of pancreatic endocrine cells could be envisaged.     

A symmetry-related sequence-structure relation of proteins

Proteins have regular tertiary structures but irregular amino acid sequences. This made it very difficult to decode the structural information in the protein sequences. Here we demonstrate that many small α protein domains have hidden sequence symmetries characteristic of their pseudo-symmetric tertiary structures. We also present a modified method of recurrent plot to reveal this kind of the hidden sequence symmetry. The results may enable us to understand part of the relations between protein sequences and their tertiary structures.     

Requirement of the Drosophila raf homologue for torso function

In Drosophila the correct formation of the most anterior and posterior regions of the larva, acron and telson is dependent on the maternally expressed terminal class of genes. In their absence, the anterior head skeleton is truncated and all the structures posterior to the abdominal segment seven are not formed. The protein predicted to be encoded by one of these genes, torso (tor), seems to be a transmembrane protein with an extracytoplasmic domain acting as a receptor and a cytoplasmic domain containing tyrosine kinase activity. Here we report that another member of the terminal-genes class, l(1)polehole (l(1)ph), which is also zygotically expressed, is the Drosophila homologue of the v-raf oncogene and encodes a potential serine-and-threonine kinase. We also show that functional l(1)ph gene product is required for the expression of a gain-of-function tor mutant phenotype, indicating that l(1)ph acts downstream of tor. Together, these results support the idea that the induction of terminal development occurs through a signal transduction system, involving the local activation of the tor-encoded tyrosine kinase at the anterior and posterior egg poles, resulting in the phosphorylation of the l(1)ph gene product. In turn, downstream target proteins may be phosphorylated, ultimately leading to the regionalized expression of zygotic target genes. Such a process is in agreement with the finding that both tor and l(1)ph messenger RNAs are evenly distributed.     

Evolutionary information for specifying a protein fold

Classical studies show that for many proteins, the information required for specifying the tertiary structure is contained in the amino acid sequence. Here, we attempt to define the sequence rules for specifying a protein fold by computationally creating artificial protein sequences using only statistical information encoded in a multiple sequence alignment and no tertiary structure information. Experimental testing of libraries of artificial WW domain sequences shows that a simple statistical energy function capturing coevolution between amino acid residues is necessary and sufficient to specify sequences that fold into native structures. The artificial proteins show thermodynamic stabilities similar to natural WW domains, and structure determination of one artificial protein shows excellent agreement with the WW fold at atomic resolution. The relative simplicity of the information used for creating sequences suggests a marked reduction to the potential complexity of the protein-folding problem.     

Nir2碳端结构域的晶体结构

Nir2蛋白具有多个结构域，在生物体中可能发挥多种生理功能。本实验用MAD软件对大肠杆菌BL21（plys）原核表达的Nir2蛋白碳端进行了晶体结构分析。结果显示，硒蛋氨酸取代蛋白晶体及未取代蛋白晶体同属空间群P212121,晶胞参数a=109.468 Å, b=120.621 Å, c=70.315 Å，分辨率分别为2.2 Å和2.7 Å。一个不对称单位含有三个分子，含水量为48%。Nir2蛋白碳端由C片层和N片层构成，其中C片层与钙ATPase的催化区域P相似，N片层为许多不相关的功能性蛋白所共有。研究证明二者之间的静电作用是影响Nir2蛋白碳端与Pyk2 FERM区域结合的主要因素。该晶体结构的分析结果为进一步研究Nir2蛋白碳端的潜在功能提供了结构基础。     

石斛兰SIZ1基因的克隆及序列分析

首次从石斛兰中克隆到1个类SIZ1基因,命名为DenSIZ1, 运用生物信息学软件分析与预测其蛋白的理化性质、结构组成、二级结构、功能结构域与亚细胞定位．结果表明,DenSIZ1可能属于PIAS家族SUMO E3连接酶,包括3个重要的功能结构域SAP、PHD和zf-MIZ,且该蛋白可能定位于细胞核中,与PIAS家族有较高的同源性．