龋齿致病菌变形链球菌蛋白 Smu.260 的 制备、结晶及初级晶体学分析

变形链球菌 (Streptococcus mutans) 是最主要的龋齿致病菌,其基因 Smu.260 编码一个约 23 ku (200 个氨基酸 ) 的蛋白质. Smu.260的 DNA 片段被克隆到表达载体 pET28a 后在大肠杆菌 BL21(DE3) 菌株中表达得到很好的产量. 产物 Smu.260 蛋白通过 Ni²⁺亲和柱和分子筛两步法纯化,并发现纯化后的蛋白以两种形式存在,二聚体 (约46 ku) 和四聚体,前者呈亮黄色,后者无色. 采用悬滴气象扩散法得到了二聚体形式的晶体. 晶体的 X 射线衍射分辨率达到 2.3埃,晶体属正交空间群 P2₁2₁2₁,晶格参数为a=89.88埃, b=90.91埃, c=105.17埃. 晶胞不对称单元内估计含有一个二聚体,溶剂含量为 53％ .     

灵芝免疫调节蛋白LZ-8的制备和晶体分析

LZ-8蛋白是从灵芝菌丝中分离到的真菌免疫调节蛋白,具有多种免疫调节功能,然而这一蛋白的作用机制尚不清楚。通过蛋白质晶体结构的解析,能够得到蛋白质空间结构特点,从而阐述蛋白质功能的机制。旨在得到LZ-8蛋白的晶体,并获得空间结构数据。以pET21a为表达载体,获得诱导表达的rLZ-8,通过亲和层析、分子筛凝胶层析和阴离子交换层析纯化,蛋白纯度在98%以上,采用悬滴气相扩散法得到蛋白晶体,并获得3.2?数据,为进一步对真菌免疫调节蛋白功能和结构的研究奠定了基础。     

巨大芽孢杆菌WSH-002丙氨酸脱氢酶066晶体制备及X-射线衍射分析

丙氨酸脱氢酶可逆催化丙氨酸脱氨生成丙酮酸,在氨基酸和酮酸的合成及代谢中至关重要.本研究通过PCR从巨大芽孢杆菌WSH-002中克隆并构建了丙氨酸脱氢酶基因(aldBM066)的原核表达载体,经原核表达后,采用Ni-NTA亲和层析法和阴离子交换色谱法纯化获得蛋白AldBM066,在289 K下以座滴法进行晶体生长条件筛选和制备.通过对蛋白质结晶条件的筛选,最终在蛋白质浓度为15 mg/mL及含有0.1 mol/L 乙酸钠（pH 5.0）和2.4 mol/L甲酸钠的缓冲液中获得了理想的蛋白质晶体,晶体大小约为210 μm×180 μm×150 μm,X-射线衍射数据显示,该蛋白质晶体衍射分辨率为2.88 A,空间群为三方晶系,晶胞参数为a=b=118.71 A,c=150.51 A,α=β=90°,γ=120°,每个不对称单位中含有1个AldBM066单体,马修斯系数为2.62 A3/Da,溶剂含量约为53.02%.衍射数据的成功收集为解析巨大芽孢杆菌WSH-002中丙氨酸脱氢酶的三维结构奠定了前期基础,将有助于阐明以单体存在的丙氨酸脱氢酶的催化机制.     

福氏志贺菌硫氧还过氧化物酶的晶体生长和初步晶体学研究（英文）

过氧化物酶是一类广泛存在于生物体内的抗氧化剂,清除有氧代谢过程中产生的活性氧,对于保护机体内的生物大分子有重要的生物学功能.福氏志贺菌硫氧还过氧化物酶(SF2523)作为过氧化物酶家族的一员,通过清除福氏志贺菌体内的活性氧,在维持其活性和致病性上起重要作用.目前,SF2523的三维结构还没有得到解析,其具体的功能机制也尚不清楚.为了得到SF2523蛋白的三维结构,进而了解具体的功能机制,实验获得了均一稳定的可溶蛋白,验证具有体外活性,培养出可用于X射线衍射的蛋白质晶体.在中国科学院高能物理研究所同步辐射装置收到晶体的衍射数据供结构解析使用.SF2523晶体属于空间群P2_12_12_1,晶胞参数为a=35.80,b=50.63,c=88.52,α=β=γ=90.00°,每个晶体学不对称单位含有1个蛋白质分子,马修斯系数为2.03~3/u,溶剂含量为39.56%.     

巨大芽孢杆菌WSH-002丙氨酸脱氢酶066晶体制备及X-射线衍射分析（英文）

丙氨酸脱氢酶可逆催化丙氨酸脱氨生成丙酮酸,在氨基酸和酮酸的合成及代谢中至关重要.本研究通过PCR从巨大芽孢杆菌WSH-002中克隆并构建了丙氨酸脱氢酶基因(aldBM066)的原核表达载体,经原核表达后,采用Ni-NTA亲和层析法和阴离子交换色谱法纯化获得蛋白AldBM066,在289 K下以座滴法进行晶体生长条件筛选和制备.通过对蛋白质结晶条件的筛选,最终在蛋白质浓度为15 mg/mL及含有0.1 mol/L乙酸钠(p H 5.0)和2.4 mol/L甲酸钠的缓冲液中获得了理想的蛋白质晶体,晶体大小约为210μm×180μm×150μm,X-射线衍射数据显示,该蛋白质晶体衍射分辨率为2.88,空间群为三方晶系,晶胞参数为a=b=118.71,c=150.51,α=β=90°,γ=120°,每个不对称单位中含有1个AldBM066单体,马修斯系数为2.623/Da,溶剂含量约为53.02%.衍射数据的成功收集为解析巨大芽孢杆菌WSH-002中丙氨酸脱氢酶的三维结构奠定了前期基础,将有助于阐明以单体存在的丙氨酸脱氢酶的催化机制.     

应用蛋白质组学技术筛选胃癌SGC-7901细胞中的let-7a功能相关蛋白

为探讨let-7a表达下调在胃癌发病中的机制,高通量地检测了与let-7a功能相关的蛋白质.首先采用基因克隆技术稳定过表达SGC-7901细胞系的let-7a基因,然后用蛋白质组学技术研究稳定过表达该基因对SGC-7901细胞蛋白质表达谱的影响.通过对SGC-7901/let-7a细胞的蛋白质表达谱改变的研究,并用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析鉴定了10个差异表达蛋白质.这些差异表达蛋白质可能是let-7a功能相关蛋白质,其中抗氧化蛋白2、胰岛素样生长因子结合蛋白2、二硫化蛋白异构酶A2、四氢叶酸合成酶、细胞周期素依赖性激酶抑制蛋白1、Rho-GTPsae激活蛋白4表达上调,Skp2蛋白、血小板黏附蛋白CD41、纤维连接蛋白、Cks1蛋白表达下调.部分差异表达蛋白质如细胞周期素依赖性激酶抑制蛋白1、Skp2蛋白和纤维连接蛋白经蛋白质印迹分析进行了验证.在SGC-7901/let-7a中鉴定的10个差异表达蛋白质涉及到细胞周期的调控、分子基因表达调控、细胞黏附、细胞代谢等众多事件,它们可能作为let-7a功能相关蛋白质,为阐明let-7a表达下调在胃癌发病中的机制提供了重要线索.     

龋齿致病菌变性链球菌蛋白Smu_195c的制备、结晶及初级晶体学分析

变性链球菌(Streptococcus mutans)是最主要的龋齿致病菌.其中Smu_195c为一个约10ku(86个氨基酸)的蛋白质,序列比对结果显示,它没有包含保守的结构域,功能也是未知的.为揭示其功能,在大肠杆菌BL21(DE3)中表达了N端结合了6个His的编码Smu_195c的DNA片段.通过悬滴法得到了两种不同晶系的晶体.第一种属于空间群P6122或P6522,晶格参数为a=b=62.93!,c=90.63",γ=120°;第二种属于空间群P41212或P43212,晶格参数为a=b=57.97#,c=103.51$.N端带有His的蛋白质长出的晶体属于第一种,而切掉N端的6个His后的蛋白质长出的晶体两种都有.     

福氏志贺菌硫氧还过氧化物酶的晶体生长和初步晶体学研究

过氧化物酶是一类广泛存在于生物体内的抗氧化剂,清除有氧代谢过程中产生的活性氧,对于保护机体内的生物大分子有重要的生物学功能。福氏志贺菌硫氧还过氧化物酶(SF2523)作为过氧化物酶家族的一员,通过清除福氏志贺菌体内的活性氧,在维持其活性和致病性上起重要作用。目前,SF2523的三维结构还没有得到解析,其具体的功能机制也尚不清楚。为了得到SF2523蛋白的三维结构,进而了解具体的功能机制,实验获得均一稳定的可溶蛋白,验证具有体外活性,培养出可用于X射线衍射的蛋白质晶体。在中国科学院高能物理研究所同步辐射收到晶体的衍射数据供结构解析使用。SF2523晶体的属于空间群P2₁2₁2₁,晶胞参数为a = 35.80 ?, b = 50.63 ?, c = 88.52 ?, α = β = γ = 90.00°,每个晶体学不对称单位含有1个蛋白质分子,马修斯系数为2.03 ?₃ /u,溶剂含量为39.56%。     

假坚强芽孢杆菌四氢嘧啶羟化酶的晶体制备及X-射线衍射研究

摘要:【目的】假坚强芽孢杆菌四氢嘧啶羟化酶蛋白纯化、晶体制备及X-射线衍射研究。【方法】通过PCR从假坚强芽孢杆菌OF4中克隆获得四氢嘧啶羟化酶基因,构建原核表达载体,经过原核表达,采用Ni-NTA亲和层析法和分子排阻色谱法纯化蛋白,289 K下采用座滴法进行晶体筛选和制备,在低温100 K下通过X-射线衍射仪(Rigaku MicroMax-007 HF)收集晶体衍射数据。【结果】通过原核表达及纯化成功获得了适合晶体生长的蛋白BpEctD。通过筛选最终在蛋白浓度为6.5 mg/mL及含有0.2 mol/L MgCl2·6H2O,0.1 mol/L Bis-Tris pH6.5,25% (W/V) 聚乙二醇3,350的缓冲液中获得了理想的蛋白晶体,其大小约为360 μm×240μm×60 μm,并在100K下成功收集了衍射数据,晶体衍射分辨率为2.40,空间群为三斜晶系P1,晶胞参数为a=45.18,b=58.87,c=68.81,α=77.48°,β=86.03°,γ=66.97°,每个不对称单位中含有2 个BpEctD单体,马修斯系数为2.44 3/Da,溶剂含量约为49.53%。【结论】衍射数据的成功收集为假坚强芽孢杆菌OF4四氢嘧啶羟化酶三维结构的解析奠定了前期基础,将有助于阐明四氢嘧啶羟化酶的催化机制。     

富硒螺旋藻中含硒藻蓝蛋白的纯化、结晶及初步晶体学研究

从富硒螺旋藻中提取含硒藻蓝蛋白,经凝胶色谱和离子交换色谱纯化,应用悬滴气相扩散法,采用(NH4)2SO4和PEG4000作沉淀剂,获得了该蛋白质晶体的两种晶型.晶型Ⅰ属于单斜晶系,晶胞参数a=10.80 nm,b=11.70 nm,c=18.40 nm,β=90.2°,晶体空间群属于P21.单位晶胞中每个晶体学不对称单位含12个(αβ)单体,晶体衍射的最高分辨率达0.28 nm.晶型Ⅱ为六方晶系,晶胞参数为a=b=15.5 nm,c=4.03 nm,晶体空间群属于P63.晶体衍射的最高分辨率达0.28 nm.单位晶胞中每个晶体学不对称单位含1个(αβ)单体.对分子在晶体中的可能堆积方式进行了讨论.     

Protein preparation, crystallization and preliminary X-ray crystallographic analysis of Smu.1475c from caries pathogen Streptococcus mutans

The gene smu.1475c encodes a putative protein of 211 residues in Streptococcus mutans, a primary pathogen for human dental caries. In this work, smu.1475c was cloned into pET28a and expressed in good amount from the E. coli strain BL21 (DE3). Smu.1475c protein was purified to homogeneity in a two-step procedure of Ni2+ chelating and size exclusion chromatography. Crystals were obtained by hanging-drop vapor-diffusion method and diffracted to 2.7 angstroms resolution. The crystal belongs to orthorhombic space group P2(1)2(1)2(1) with cell dimension of a = 68.3 angstroms, b = 105.9 angstroms, c = 136.2 angstroms. The asymmetric unit is expected to contain four molecules with solvent content of 49.4%.     

A Novel Cytotoxic Cerium Complex: Aquatrichloridobis(1,10‐phenanthroline)cerium(III) (KP776). Synthesis,Characterization, Behavior in H2O,Binding towards Biomolecules,and Antiproliferative Activity

The lanthanide complex aquatrichloridobis(1,10‐phenanthroline)cerium(III) [Ce(phen)₂(H₂O)Cl₃] (KP776) was fully characterized by elemental analysis, IR‐, and ¹H‐ and ¹³C‐NMR spectroscopy, as well as TG/DTA measurements, and its behavior in H₂O, important for the application as a chemotherapeutic, was studied. In addition, the binding of KP776 to nucleotides and single serum proteins was investigated by capillary electrophoresis, whereas binding to proteins in human plasma was observed by ICP‐MS. The compound shows promising anticancer properties in vitro: proliferation of human cancer cell lines is strongly inhibited with IC₅₀ values in the very low micromolar range.     

LuxS-mediated signaling in Streptococcus mutans is involved in regulation of acid and oxidative stress tolerance and biofilm formation

LuxS-mediated quorum sensing has recently been shown to regulate important physiologic functions and virulence in a variety of bacteria. In this study, the role of luxS of Streptococcus mutans in the regulation of traits crucial to pathogenesis was investigated. Reporter gene fusions showed that inactivation of luxS resulted in a down-regulation of fructanase, a demonstrated virulence determinant, by more than 50%. The LuxS-deficient strain (TW26) showed increased sensitivity to acid killing but could still undergo acid adaptation. Northern hybridization revealed that the expression of RecA, SmnA (AP endonuclease), and Nth (endonuclease) were down-regulated in TW26, especially in early-exponential-phase cells. Other down-regulated genes included ffh (a signal recognition particle subunit) and brpA (biofilm regulatory protein A). Interestingly, the luxS mutant showed an increase in survival rate in the presence of hydrogen peroxide (58.8 mM). The luxS mutant formed less biofilm on hydroxylapatite disks, especially when grown in biofilm medium with sucrose, and the mutant biofilms appeared loose and hive-like, whereas the biofilms of the wild type were smooth and confluent. The mutant phenotypes were complemented by exposure to supernatants from wild-type cultures. Two loci, smu486 and smu487, were identified and predicted to encode a histidine kinase and a response regulator. The phenotypes of the smu486 smu487 mutant were, in almost all cases, similar to those of the luxS mutant, although our results suggest that this is not due to AI-2 signal transduction via Smu486 and Smu487. This study demonstrates that luxS-dependent signaling plays critical roles in modulating key virulence properties of S. mutans.     

The zebrafish slow-muscle-omitted gene product is required for Hedgehog signal transduction and the development of slow muscle identity

Hedgehog proteins mediate many of the inductive interactions that determine cell fate during embryonic development. Hedgehog signaling has been shown to regulate slow muscle fiber type development. We report here that mutations in the zebrafish slow-muscle-omitted (smu) gene disrupt many developmental processes involving Hedgehog signaling. smu(-/-) embryos have a 99% reduction in the number of slow muscle fibers and a complete loss of Engrailed-expressing muscle pioneers. In addition, mutant embryos have partial cyclopia, and defects in jaw cartilage, circulation and fin growth. The smu(-/-) phenotype is phenocopied by treatment of wild-type embryos with forskolin, which inhibits the response of cells to Hedgehog signaling by indirect activation of cAMP-dependent protein kinase (PKA). Overexpression of Sonic hedgehog (Shh) or dominant negative PKA (dnPKA) in wild-type embryos causes all somitic cells to develop into slow muscle fibers. Overexpression of Shh does not rescue slow muscle fiber development in smu(-/-) embryos, whereas overexpression of dnPKA does. Cell transplantation experiments confirm that smu function is required cell-autonomously within the muscle precursors: wild-type muscle cells rescue slow muscle fiber development in smu(-/-) embryos, whereas mutant muscle cells cannot develop into slow muscle fibers in wild-type embryos. Slow muscle fiber development in smu mutant embryos is also rescued by expression of rat Smoothened. Therefore, Hedgehog signaling through Slow-muscle-omitted is necessary for slow muscle fiber type development. We propose that smu encodes a vital component in the Hedgehog response pathway.     

Crystal structure of chicken liver basic fatty acid-binding protein at 2.7 Å resolution

The three-dimensional structure of chicken liver basic fatty acid-binding protein has been determined at 2.7 Å resolution by X-ray crystallography. Phases were calculated using the multiple isomorphous replacement procedure and a preliminary model was built. This model, with an initial R-factor of 0.57, was then improved by a cycle of refinement by simulated annealing which brought the R factor down to 0.32. The protein is structured as a compact 10-stranded--barrel which encapsulates a residual electron density that can be interpreted as a fatty acid molecule. The NH₂-terminus portion of the molecule contains two short -helices. The structure of this liver protein appears very similar to that of the Escherichia coli derived rat intestinal FABP recently determined by X-ray diffraction methods.     

14-3-3 Binding to ataxin-1(ATXN1) regulates its dephosphorylation at Ser-776 and transport to the nucleus

Spinocerebellar ataxia type 1 (SCA1) is a lethal neurodegenerative disorder caused by expansion of a polyglutamine tract in ATXN1. A prominent site of pathology in SCA1 is cerebellar Purkinje neurons where mutant ATXN1 must enter the nucleus to cause disease. In SCA1, phosphorylation of ATXN1 at Ser-776 modulates disease. Interestingly, Ser-776 is located within a region of ATXN1 that harbors several functional motifs including binding sites for 14-3-3, and splicing factors RBM17 and U2AF65. The interaction of ATXN1 with these proteins is thought to be regulated by the phosphorylation status of Ser-776. In addition, Ser-776 is adjacent to the NLS in ATXN1. Although pS776-ATXN1 is enriched in nuclear extracts of cerebellar cells, the vast majority of 14-3-3 is in the cytoplasmic fraction. We found that dephosphorylation of cytoplasmic pS776-ATXN1 is blocked by virtue of it being in a complex with 14-3-3. In addition, data suggest that binding of 14-3-3 to cytoplasmic ATXN1 impeded its transport to the nucleus, suggesting that 14-3-3 must disassociate from ATXN1 for transport of ATXN1 to the nucleus. Consistent with this hypothesis is the observation that once in the nucleus pS776 is able to be dephosphorylated. Evidence is presented that PP2A is the pS776-ATXN1 phosphatase in the mammalian cerebellum. In the nucleus, we propose that dephosphorylation of pS776-ATXN1 by PP2A regulates the interaction of ATXN1 with the splicing factors RBM17 and U2AF65.     

Functional role of oxygen-containing residues in the fifth transmembrane segment of the Na,K-ATPase alpha subunit

The functional roles of Tyr771, Thr772, and Asn776 in the fifth transmembrane segment of the Na, K-ATPase alpha subunit were studied using site-directed mutagenesis, expression, and kinetics analysis. Nonconservative replacements Thr772Tyr and Asn776Ala led to reduced Na,K-ATPase turnover. Replacements at these positions (Asn776Ala, Thr772Leu, and Thr772Tyr) also led to high Na-ATPase activity (in the absence of K+). However, Thr772- and Asn776-substituted enzymes showed only small alterations in the apparent Na+ and K+ affinities (K1/2 for Na,K-ATPase activation). Thus, the high Na-ATPase activity does not appear related to cation-binding alterations. It is probably associated with conformational alterations which lead to an acceleration of enzyme dephosphorylation by Na+ acting at the extracellular space (Argüello et al. J. Biol. Chem. 271, 24610-24616, 1996). Nonconservative substitutions at position 771 (Tyr771Ala and Tyr771Ser) produced a significant decrease of enzyme turnover. Enzyme-Na+ interaction was greatly changed in these enzymes, while their activation by K+ did not appear affected. Although the Na+ K1/2 for Na,K-ATPase stimulation was unchanged (Tyr771Ala, Tyr771Ser), the activation by this cation showed no cooperativity (Tyr771Ala, nHill = 0.75; Tyr771Ser, nHill = 0.92; Control, nHill = 2.28). Substitution Tyr771Phe did not lead to a significant reduction in the cooperativity of the ATPase Na+ dependence (nHill = 1.91). All Tyr771-substituted enzymes showed low steady-state levels of phosphoenzyme during Na-activated phosphorylation by ATP. Phosphorylation levels were not increased by oligomycin, although the drug bound and inactivated Tyr771-substituted enzymes. No E1 left and right arrow E2 equilibrium alterations were detected using inhibition by vanadate as a probe. The data suggest that Tyr771 might play a central role in Na+ binding and occlusion without participating in K+-enzyme interactions.     

Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket

Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp, Gln, Ala), Asp804 (Glu, Asn, Ala), and Asp808 (Glu, Asn, Ala). Electrogenic cation transport properties of these 12 mutants were analyzed in two-electrode voltage-clamp experiments on Xenopus laevis oocytes by measuring the voltage dependence of K+-stimulated stationary currents and pre-steady-state currents under electrogenic Na+/Na+ exchange conditions. Whereas mutants D804N, D804A, and D808A hardly showed any Na+/K+ pump currents, the other constructs could be classified according to the [K+] and voltage dependence of their stationary currents; mutants N776A and E779Q behaved similarly to the wild-type enzyme. Mutants E779D, E779A, D808E, and D808N had in common a decreased apparent affinity for extracellular K+. Mutants N776Q, N776D, and D804E showed large deviations from the wild-type behavior; the currents generated by mutant N776D showed weaker voltage dependence, and the current-voltage curves of mutants N776Q and D804E exhibited a negative slope. The apparent rate constants determined from transient Na+/Na+ exchange currents are rather voltage-independent and at potentials above -60 mV faster than the wild type. Thus, the characteristic voltage-dependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn776 and Asp804, respectively, decreases the extracellular Na+ affinity.