Complementary Roles of Two DNA Protection Proteins from Deinococcus geothermalis

The roles of two interrelated DNA protection protein in starved cells (Dps)—putative Dps Dgeo_0257 and Dgeo_0281—as orthologous proteins to DrDps1 for DNA binding, protection, and metal ion sensing were characterised in a Deinococcus geothermalis strain. Dgeo_0257 exhibited high DNA-binding affinity and formed a multimeric structure but lacked the conserved amino acid sequence for ferroxidase activity. In contrast, the Dgeo_0281 (DgDps1) protein was abundant in the early exponential phase, had a lower DNA-binding activity than Dgeo_0257, and was mainly observed in its monomeric or dimeric forms. Electrophoretic mobility shift assays demonstrated that both purified proteins bound nonspecifically to DNA, and their binding ability was affected by certain metal ions. For example, in the presence of ferrous and ferric ions, neither Dgeo_0257 nor Dgeo_0281 could readily bind to DNA. In contrast, both proteins exhibited more stable DNA binding in the presence of zinc and manganese ions. Mutants in which the dps gene was disrupted exhibited higher sensitivity to oxidative stress than the wild-type strain. Furthermore, the expression levels of each gene showed an opposite correlation under H₂O₂ treatment conditions. Collectively, these findings indicate that the putative Dps Dgeo_0257 and DgDps1 from D. geothermalis are involved in DNA binding and protection in complementary interplay ways compared to known Dps.     

Determination of Site Specific Adsorption Energies of CO on Copper

The binding energies of (isolated) CO molecules adsorbed at several atomic sites (terrace, step, kink) on a number of differently oriented copper surfaces have been measured by thermal desorption spectroscopy (TDS). In addition to the three low-indexed Cu surfaces several regular stepped and kinked single crystal surfaces have been employed. Using LEED measurements together with available data in the literature allowed identification of the various different CO adlayers and to assign the different TDS binding energies to the different adsorbate sites. For the close-packed surfaces binding energies between 47 kJ/mol (Cu(111)) and 51 kJ/mol (Cu(100)) were observed, which increased to 58 kJ/mol for CO molecules bound to step edges. Unexpectedly, for kink sites the same binding energy (to within 1 kJ/mol) as for step edges was observed. Moreover, a very similar binding energy of 58 kJ/mol was also measured for random defect sites on sputtered and on poly-crystalline substrates.     

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K

The catalytic contribution of His48 in the active site of porcinepancreatic phospholipase A2 was examined using site-directedmutagenesis. Replacement of His48 by lysine (H48K) gives riseto a protein having a distorted lipid binding pocket. Activityof this variant drops below the detection limit which is 10⁷-foldlower than that of the wild-type enzyme. On the other hand,the presence of glutamine (H48Q) or asparagine (H48N) at thisposition does not affect the structural integrity of the enzymeas can be derived from the preserved lipid binding propertiesof these variants. However, the substitutions H48Q and H48Nstrongly reduce the turnover number, i.e. by a factor of 10⁵.Residual activity is totally lost after addition of a competitiveinhibitor. We conclude that proper lipid binding on its ownaccelerates ester bond hydrolysis by a factor of 10². With theselected variants, we were also able to dissect the contributionof the hydrogen bond between Asp99 and His48 on conformationalstability, being 5.2 kJ/mol. Another hydrogen bond with His48is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycolinteracts with the enzyme. Its contribution to binding of theinhibitor in the presence of an interface was found to be 5.7kJ/mol.     

生物信息学方法筛选华支睾吸虫成虫功能基因——钙调神经磷酸酶B亚基样蛋白

目的本文通过建立华支睾吸虫成虫cDNA文库,筛选其功能基因。方法应用SMART方法构建华支睾吸虫成虫cDNA文库,进行大量EST测序,然后应用生物信息学方法将EST序列与GenBank中登陆的序列进行同源性比对、序列拼接及基因完整性判断;并应用NCBI上的RPSBLAST对所筛选基因的保守域进行搜索比对,利用PredictProtein分析、预测其功能域及二级结构。结果从华支睾吸虫成虫cDNA文库中筛选的钙调神经磷酸酶B亚基样蛋白(CsCBLP)基因,经同源性分析,CsCBLP与褐家鼠钙调神经磷酸酶(CaN)同源性为53%,与尾刺耐格里原虫CaNB同源性为40%,与新小杆线虫属结合蛋白的同源性为51%;保守域的比对及功能域、二级结构的预测显示所筛选的CsCBLP是钙调神经磷酸酶B亚基的的类似物,属于钙结合蛋白家族。结论应用生物信息学方法从华支睾吸虫成虫cDNA文库中筛选出钙调神经磷酸酶B亚基样蛋白基因。     

Selective DNA‐Binding by Designed Bisbenzamidine‐Homeodomain Chimeras

We report the construction of conjugates between three variants of the helix 3 region of a Q50K engrailed homeodomain and bisbenzamidine minor‐groove DNA binders. The hybrid featuring the sequence of the native protein failed to bind to DNA; however, modifications that increased the α‐helical folding propensity of the peptide allowed specific DNA binding by a bipartite (major/minor groove) interaction.     

Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A- C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed.      

Stabilization of bzip peptides through incorporation of fluorinated aliphatic residues

Two fluorinated amino acids, 5,5,5-trifluoroisoleucine (5TFI) and (2S,3R)-4,4,4-trifluorovaline (4TFV), which have been shown to serve as isoleucine surrogates in protein synthesis in Escherichia coli, have been incorporated in vivo into basic leucine zipper (bzip) peptides derived from GCN4. The extents of residue-specific incorporation of 5TFI and 4TFV were 90 and 88 %, respectively, of the encoded isoleucine residues, as evidenced by MALDI mass spectrometry and amino acid analysis. Both circular dichroism and equilibrium sedimentation studies of the fluorinated bzip peptides indicated preservation of secondary and higher-order protein structure. Thermal-denaturation experiments showed an increase of 27 degrees C in melting temperature when isoleucine was replaced by 5TFI. However, the T(m) of the peptide containing 4TFV was increased by only 4 degrees C over that of the peptide containing valine. Similar trends were observed in chemical denaturation studies in which DeltaDeltaG(unfold) in water was determined to be 2.1 or 0.3 kcal mol(-1) upon incorporation of 5TFI or 4TFV, respectively. When the fluorinated peptides were tested for DNA binding, both their affinity and specificity were similar to those of the respective hydrogenated peptides. These results suggest that fluorinated amino acids, even when introduced into the same positions, can have markedly different effects on the physical properties of proteins, while having little impact on secondary and higher-order structure.     

A Glutathione‐Responsive Short Sequence of Metal–Organic Complex Array

A short metal–organic complex array (MOCA) containing a sequence of RPtRRu ( 1_Cl ) was found to exhibit unique responses to a major biothiol, glutathione (GSH). Upon binding of GSH to 1_Cl , the resultant 1:1 complex ( 1_GS ) formed nanofibrous assemblies that suggested supramolecular polymerization through the double‐salt‐bridge structure formation. The binding behavior of this MOCA sequence to calf thymus DNA was also dependent on GSH; a larger conformational change of DNA was observed upon binding with 1_GS , relative to that with 1_Cl .     

Metal-dependent stabilization of an active HMG protein

Using a cloned single domain of the high mobility group protein1 (HMGB1), we evaluated the effect of introducing metal bindingsite(s) on protein stability and function. An HMG domain isa conserved sequence of 80 amino acids rich in basic, aromaticand proline residues that is active in binding DNA in a sequence-or structure-specific manner. The design strategy focuses onanchoring selected regions of the protein, specifically loopsand turns in the molecule, using His–metal ligands. Changesin secondary structure, thermostability and DNA binding propertiesof a series of such mutants were evaluated. The two most stablemutant constructs contain three surface histidine replacements(two metal binding sites) in the regions encompassing both turnsof the molecule. On ligation with the divalent nickel cation,the stability of these two triple histidine mutants (I38H/N51H/D55Hand G39H/N51H/D55H) increases by 1.3 and 1.6 kcal/mol, respectively,relative to the wild-type protein, although the creation ofbinding sites per se destabilizes the protein. The DNA-bindingproperties of the modified proteins are not impaired by theintroduction of the metal binding motifs. These results indicatethat it is feasible to stabilize protein tertiary structureusing appropriate placement of surface His–metal bondswithout loss of function.     

牙本质基质蛋白1功能片段的融合表达及其胞内定位

目的构建含单核苷酸多态性(Single nucleotide polymorphism,SNP)位点不同碱基的37 kD的N-牙本质基质蛋白1(Dentin matrix protein 1,DMP1)和全长DMP1基因重组表达质粒,并分析重组蛋白在HEK293细胞中的表达及定位。方法利用定点突变改造DMP1基因,获得DMP1基因的SNP rs10019009的不同基因型,将含SNP位点不同碱基的37 kD N-DMP1和全长DMP1基因定向克隆入含增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)的质粒pcDNA3.1-EGFP中,构建重组质粒pcDNA3.1-DMP1-EGFP,通过脂质体法瞬时转染HEK293细胞,荧光显微镜观察重组融合蛋白DMP1-EGFP的表达及胞内定位。结果 DNA测序结果表明,定点突变后的DMP1基因的碱基序列与设计序列完全一致;PCR和DNA测序显示重组质粒pcDNA3.1-DMP1-EGFP构建正确;融合蛋白DMP1-EGFP在HEK293细胞中主要表达于细胞胞浆中。结论成功构建了含rs10019009-SNP位点不同碱基的37 kD的N-DMP1和全长DMP1基因重组表达质粒,并在HEK293细胞的胞浆中有效表达,为进一步研究DMP1基因的功能奠定了基础。     

光谱法研究迷迭香酸和牛血清白蛋白的相互作用

利用荧光和圆二色光谱研究了迷迭香酸(RA)与牛血清白蛋白(BSA)之间的相互作用.通过荧光猝灭测得在301、308和315 K时,RA与BSA的结合常数K分别为4.18×10~4、3.62×10~4和2.52×10~4 L/mol,表明RA与BSA间具有较强的结合作用,属于静态猝灭.热力学参数计算结果表明RA与BSA相互作用力以范德华力及氢键作用力为主.圆二色光谱、红外及拉曼光谱、荧光同步光谱研究表明相互作用后BSA的二级结构发生微小变化.此外,常见金属离子对结合有较为显著的影响.     

Integration Host Factor Binds DNA Holliday Junctions

Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg²⁺ but accelerates the transition rate at 10 mM Mg²⁺. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.     

Stability of aspartate aminotransferase from Sulfolobus solfataricus

Aspartate aminotransferase from Sulfolobus solfataricus (SsAspAT) is an extremely thermophilic and thermostable dimeric enzyme which retains its structure and reaches maximal activity at 100 degrees C. The structural stability of this protein was investigated by coupling isothermally and thermally induced denaturation studies to molecular modeling. Gel filtration analysis indicated that SsAspAT unfolds with an N2 reversible 2D mechanism. In the molecular model, a cluster of hydrophobic residues was shown at the interface between the subunits of SsAspAT and suggested this cluster as a structural feature stabilizing the enzyme quaternary structure. At 25 degrees C, SsAspAT is less resistant to guanidinium chloride-induced denaturation than the cytosolic aspartate aminotransferase from pig heart (cpAspAT), which was chosen as a mesophilic counterpart in the thermodynamic analysis since it shares with SsAspAT the two-state unfolding mechanism. Therefore, in the case of aspartate aminotransferases, thermal stability does not correlate with the stability against chemical denaturants. Isothermal denaturation curves at 25 degrees C and melting profiles recorded in the presence of guanidinium chloride showed that the delta G degrees (H2O) at 25 degrees C of SsAspAT exceeds that of cpAspAT by roughly 15 kJ/mol; the parameter delta n, related to the number of binding sites for the denaturant differentially exposed in unfolded and folded states, is higher for SsAspAT than for cpAspAT; and delta Cp is lower for the thermophilic enzyme than for the mesophilic one by 8 kJ/K.mol. These results are indicative of a less hydrophobic core for SsAspAT than cpAspAT. In agreement with this, the molecular model predicts that some charged side chains are buried in SsAspAT and interact to form an H-bond/ion-pair network.      

Isomerization of n-hexane over nickel-loaded zeolite catalysts

The isomerization of n-hexane to branched-chain isomers was studied over various zeolite supports containing nickel between 1 to 5 wt%. NaA, NaY, CaY and Zeolon 900H were loaded with nickel by an impregnation technique. It was observed that at a nickel content of about 2.5 wt%, all the catalysts showed maximum activity for isomerization. A catalyst containing 1 wt% Ni/CaY gave the maximum selectivity among all the catalysts studied. Increasing the nickel loading beyond 2.5 wt% Ni with CaY and Zeolon 900H led to more hydrocracking. No major change in the activity and selectivity of Ni/NaA catalysts was observed beyond 2.5 wt% Ni, whereas the activity of Ni/NaY catalysts remained almost constant over the range of nickel content studied. A catalyst containing 2.5 wt% Ni on Zeolon 900H gave the maximum yield of isomers at 643°K. The apparent activation energy of the reaction was found to be 48.6 kJ/mol for 1 wt% Ni on Zeolon 900H catalyst.