NMR研究轴向配体取代对细胞色素c结构的影响

用1D和2D NMR方法归属了Im·cyt c配合物血红素环上质子和20多个氨基酸残基质子的共振峰,集中研究和表征了Im·cyt c配合物中血红素基及轴向配体周围的环境,确定了咪唑取代cyt c中Met 80轴向配体所引起容纳血红素和轴向配体的疏水腔构象的变化,配位咪唑相对于卟啉面和His 18咪唑平面的取向。     

细胞色素c突变研究进展

在简述细胞色素c的生物功能和结构特征的基础上,综述了细胞色素c突变研究的进展,重点论述了对血红素辅基(heme)的轴向配体Met80、heme所在腔的保守氨基酸残基Tyr67以及蛋白表面的保守氨基酸残基Phe82的突变研究,并对一些突变体蛋白表现出来的特殊性质给予解释。     

神经红蛋白突变体Tyr44Phe的制备、表征和稳定性研究

构建了突变体蛋白Tyr44Phe的基因, 进行了蛋白的表达、分离纯化、谱学表征和稳定性研究. 由电喷雾质谱所得突变体蛋白的分子量与理论值一致; UV-Vis吸收光谱、荧光光谱和圆二色光谱表明, Tyr44Phe的点突变虽没有改变血红素的六配位结构, 但对血红素的构象有所影响. 突变体蛋白的热、酸稳定性研究表明, 定点突变降低了血红素与蛋白肽链之间的结合力, 导致血红素易从疏水腔中脱出, 说明Tyr44对蛋白的结构稳定性起一定的作用.     

细胞色素c咪唑配合物的~1H NMR归属及构象研究

咪唑细胞色素c(Im·cyt c)是碱式细胞色素c(cyt c)及细胞色素c蛋白分子折叠过程的中间体类似物.用二维核磁共振(2D NMR)方法完成了Im·cyt c的主链(Gly-29除外)和绝大部分侧链的质子共振峰归属.归属主要采用主链指向法(MCD)进行.由得到的NOE联结,确定了Im·cyt c的二级结构,阐明了轴向配体Met80和血红素Fe间Fe-S键的断裂以及Met80被Im取代所引起的蛋白三维构象变化.同cyt c相比,Im·cyt c中的50,60及70 s a螺旋存在较大构象变动;Tyr67-Asn70和Ile75-Thr78两个月β-turn已经消失 咪唑的配位也导致了部分氨基酸残基,尤其是血红素疏水腔附近如Trp59,Tyr67,Tyr74等残基侧链的空间位置相对变化     

紫外吸收光谱法研究Asp44在稳定肌红蛋白结构中的作用

为了了解肌红蛋白Mb表面44位天冬氨酸(Asp)残基对稳定蛋白结构的影响,用聚合酶链式反应(PCR)定点突变的技术将Mb基因上的第44位天冬氨酸的密码子GAT突变成赖氨酸的密码子AAA,获得突变体D44K。突变体蛋白在大肠杆菌BL21-DE3中成功表达并且得到纯化。用紫外-可见光谱研究野生型肌红蛋白及其突变体D44K的耐热、耐酸的变性过程。结果表明,用碱性氨基酸赖氨酸(Lys)取代酸性氨基酸Asp44残基,增强了肌红蛋白耐热、耐酸能力,说明Asp44具有稳定肌红蛋白结构的作用。为进一步研究蛋白表面氨基酸对蛋白质结构、功能的影响提供重要的试验依据。     

细胞色素b5 S64X突变体对蛋白结构及稳定性的影响

为了深入了解细胞色素b₅(Cyt b₅)64位氨基酸残基(Ser64)对血红素辅基微环境及蛋白性质的影响,我们分别对Cyt b₅ Ser64进行了保守性突变(S64T)以及非保守性突变(S64K、S64N和S64H),均为亲水性氨基酸残基。对野生型细胞色素b₅及其突变体蛋白S64X(X：T,K,N或H)的热、酸、盐酸胍变性的稳定性研究表明：4个突变体蛋白的稳定性相对于野生型都大大降低了;CD光谱表明,细胞色素b₅ S64X突变体中的α-螺旋明显减少,芳香性氨基酸残基所处的肽链结构受到了影响;盐酸胍变性荧光光谱表明,Trp22周围的蛋白肽链受到了影响,Trp22暴露于水溶液的程度加大。我们认为Ser64不仅对血红素辅基有稳定作用,同时还对维持蛋白Core 1中的第5个α-螺旋结构有重要的作用,在64位引入其他氨基酸残基影响了第5个α-螺旋的结构,并通过蛋白肽链的相互作用,使得Trp22所在的Core 2结构也受到了较为明显的扰动。     

鼠脑红蛋白突变体F106L与氰根的结合作用

构建了鼠脑红蛋白(Mouse neuroglobin)的突变体F106L, 以探求近端残基对脑红蛋白血红素口袋结构的贡献. 通过溶液核磁共振方法研究了外来配体氰根离子与NgbF106L蛋白的结合作用, 结果显示, 此结合存在动力学过程, 并且NgbF106LCN 突变蛋白氰根络合物可以可逆地释放氰根离子, 并使原来的第6配体His64(E7)又结合回到血红素铁上. 研究结果揭示, G5(Phe106)残基对脑红蛋白血红素构象而言较为保守; QM/MM结构优化结果表明, 位于G5 和FG5的近端残基对蛋白结构稳定性具有重要作用, 并可调控外来配体与蛋白作用的配位平衡与热动力学性质.     

HIV-1整合酶G140A/G149A及T661/S153Y突变后的构象变化

对HIV-1整合酶(IN)野生体(WT),G140A/G149A和T66I/S153Y突变体分别进行了5 ns的分子动力学(MD)模拟,并用成簇和动力学交叉相关图(DCCM)分析了突变前后的构象变化.整体结构分析表明,突变后IN的活性口袋尺寸变化不大,T66I/S153Y突变体分子的整体运动性提高,而G140A/G149A突变体的功能loop区柔性明显上升.IN WT的方均根涨落(RMSF)模拟值与B因子实验值的较高相关性证明了柔性分析的合理性.通过成簇分析发现,IN在突变后功能loop区构象有开合运动,构象开放的概率是:体系G140A/G149A＞T66I/S153Y＞WT.最后DCCM分析结果表明,功能性分区的弱化以及DDE基序残基运动相关性的降低均有可能是突变体G140A/G149A和T66I/S153Y产生抗药性的原因.模拟结果对理解IN突变体的抗药机理以及为基于HIV-1 IN的药物分子设计提供了理论帮助.     

HIV-1整合酶G140A/G149A及T66I/S153Y突变后的构象变化

对HIV-1整合酶(IN)野生体(WT), G140A/G149A和T66I/S153Y突变体分别进行了5 ns的分子动力学(MD)模拟, 并用成簇和动力学交叉相关图(DCCM)分析了突变前后的构象变化. 整体结构分析表明, 突变后IN的活性口袋尺寸变化不大, T66I/S153Y突变体分子的整体运动性提高, 而G140A/G149A突变体的功能loop区柔性明显上升. IN WT的方均根涨落(RMSF)模拟值与B因子实验值的较高相关性证明了柔性分析的合理性. 通过成簇分析发现, IN在突变后功能loop区构象有开合运动, 构象开放的概率是: 体系G140A/G149A＞T66I/S153Y＞WT. 最后DCCM分析结果表明, 功能性分区的弱化以及DDE基序残基运动相关性的降低均有可能是突变体G140A/G149A和T66I/S153Y产生抗药性的原因. 模拟结果对理解IN突变体的抗药机理以及为基于HIV-1 IN的药物分子设计提供了理论帮助.     

Eu3+对过氧化氢酶生物催化活性的影响1

为了了解稀土微肥使植物增产的化学机理,用紫外-可见(UV-Vis)吸收光谱、同步荧光光谱和电化学方法法研究了Eu3+与过氧化氢酶(CAT)的相互作用。结果表明,由于稀土离子易与O键合,因此,Eu3+主要与CAT肽链上的氨基酸残基中的羰基氧配位,引起肽链构象的变化,而构象的变化又会诱导CAT中的血红素结构的变化。当Eu3+浓度低时,Eu3+与CAT发生相互作用能使血红素的非平面性增加,转而使血红素中活性中心Fe(III)的暴露程度增加,因此,使CAT的电化学活性和对H2O2还原的电催化活性提高。但当Eu3+的浓度高时,Eu3+会使CAT中血红素的非平面性降低,使血红素中活性中心Fe(III)的暴露程度降低,因此,降低了CAT的电化学和生物电催化活性。这说明不同浓度的Eu3+对CAT的生物活性的影响不同,所以选择适当的浓度的Eu3+对植物的生长具有促进作用。     

酸诱导拥挤条件下肌红蛋白及突变体去折叠过程

采用紫外-可见吸收光谱、同步荧光光谱和圆二色(CD)光谱法研究拥挤试剂葡聚糖70 (Dextran70)和聚蔗糖70 (Ficoll70)存在条件下, 酸诱导野生型肌红蛋白Mb(WT)及其突变体Mb(D60K)的去折叠过程. 结果显示: 在Dextran70 和Ficoll70 两种拥挤环境下, Mb(WT)的变性中点pH值由4.25 分别降低到3.78 与3.76, 拥挤试剂加入后增强了Mb(WT)的耐酸能力; 肌红蛋白60位天冬氨酸(Asp)突变为赖氨酸(Lys)后, 变性中点pH值由4.25 降低到4.19, 耐酸性比野生型肌红蛋白有所增强, Mb(D60K)在Dextran70 和Ficoll70 两种拥挤环境下变性中点pH值由4.19 分别降至3.74 和3.12. 以上实验说明肌红蛋白表面氨基酸突变和拥挤试剂的添加起到了稳定血红素微环境、芳香族氨基酸及二级结构和保护蛋白天然状态的作用.     

Interaction of Natronobacterium pharaonis phoborhodopsin (sensory rhodopsin II) with its cognate transducer probed by increase in the thermal stability

Pharaonis phoborhodopsin (ppR, also called Natronobacterium pharaonis sensory rhodopsin II) and its transducer protein, pharaonis halobacterial transducer of ppR (pHtrII), form a signaling complex, and light signals are transmitted from the sensor to the transducer by the protein-protein interaction. A truncated pHtrII(1-159) consisting of intramembrane helices (expressing amino acid residues from the first to the 159th position) and ppR form the complex in a solution containing 0.1% n-dodecyl-beta-D-maltoside. At 75-85 degrees C, the time-dependent color loss of ppR was caused by denaturation. We found that pHtrII(1-159) retarded the denaturation rate of ppR. This increase in the thermal stability was used as a probe for the binding ability in the dark. Tyr199 of ppR and Asn74 of pHtrII(1-114) were proposed as amino acid residues interacting with each other through hydrogen bonding. Then,ppR and pHtrII(1-159) mutants at these positions were prepared to examine the effect on the binding in the dark. The wild-type and Y199F mutant can bind pHtrII(1-159), suggesting that the hydrogen bonding between these specific amino acid residues may not be the only cause of the binding, but the hydrophobic interaction via phenyl ring of ppR may contribute dominantly.     

A cytochrome c variant resistant to heme degradation by hydrogen peroxide

BACKGROUND: Cytochrome c has peroxidase-like activity and can catalyze the oxidation of a variety of organic substrates, including aromatic, organosulfur and lipid compounds. Like peroxidases, cytochrome c is inactivated by hydrogen peroxide. During this inactivation the heme prosthetic group is destroyed. RESULTS: Variants of the iso-1-cytochrome c were constructed by site-directed mutagenesis and were found to be more stable in the presence of hydrogen peroxide than the wild type. No heme destruction was detected in a triple variant (Tyr67-->Phe/Asn52-->Ile/Cys102-->Thr) with the catalytic hydrogen peroxide concentration of 1 mM, even following the loss of catalytic activity, whereas both double variants Tyr67-->Phe/Cys102-->Thr and Asn52-->Ile/Cys102-->Thr showed a greater rate of peroxide-induced heme destruction than observed with the wild-type protein. CONCLUSIONS: Heme destruction and catalytic inactivation are two independent processes. An internal water molecule (Wat166) is shown to be important in the heme destruction process. The absence of a protein radical in the resistant variant suggests that the protein radical is necessary in the heme destruction process, but presumably is not involved in the reactions leading up to the protein inactivation.     

Selenium-containing enzymes in mammals: Chemical perspectives

The chemical and biochemical route to the synthesis of the 21st amino acid in living systems, selenocysteine, is described. The incorporation of this rare amino acid residue into proteins is described with emphasis on the role of monoselenophosphate as selenium source. The role of selenocysteine moiety in natural mammalian enzymes such as glutathione peroxidase (GPx), iodothyronine deiodinase (ID) and thioredoxin reductase (TrxR) is highlighted and the effect of other amino acid residues located in close proximity to selenocysteine is described. It is evident from various studies that two amino acid residues, tryptophan and glutamine, appear in identical positions in all known members of the GPx family. According to the three-dimensional structure established for bovine GPx, these residues could constitute a catalytic triad in which the selenol group of the selenocysteine is both stabilized and activated by hydrogen bonding with the imino group of the tryptophan (Trp) residue and with the amido group of the glutamine (Gln) residue. The ID enzymes, on the other hand, do not possess any Trp or Gln residues in close proximity to selenium, but contain several histidine residues, which may play important roles in the catalysis. The TrxR enzymes also possess some basic histidines, but the most important amino acid residues are the cysteines which constitute the internal cofactor systems along with the catalytically active selenocysteine. The catalytic activity and substrate specificity of all three selenoenzymes are described. The reactivity of selenocysteine residues in selenoenzymes towards metal-based drugs such as goldthioglucose is also described.     

The Catalytic Mechanism of Fluoroacetate Dehalogenase: A Computational Exploration of Biological Dehalogenation

The biological dehalogenation of fluoroacetate carried out by fluoroacetate dehalogenase is discussed by using quantum mechanical/molecular mechanical (QM/MM) calculations for a whole‐enzyme model of 10 800 atoms. Substrate fluoroacetate is anchored by a hydrogen‐bonding network with water molecules and the surrounding amino acid residues of Arg105, Arg108, His149, Trp150, and Tyr212 in the active site in a similar way to haloalkane dehalogenase. Asp104 is likely to act as a nucleophile to attack the α‐carbon of fluoroacetate, resulting in the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule to the carbonyl carbon atom. The cleavage of the strong C? F bond is greatly facilitated by the hydrogen‐bonding interactions between the leaving fluorine atom and the three amino acid residues of His149, Trp150, and Tyr212. The hydrolysis of the ester intermediate is initiated by a proton transfer from the water molecule to His271 and by the simultaneous nucleophilic attack of the water molecule. The transition state and produced tetrahedral intermediate are stabilized by Asp128 and the oxyanion hole composed of Phe34 and Arg105.     

Crystal Structure of H227A Mutant of Arginine Kinase in Daphnia magna Suggests the Importance of Its Stability

Arginine kinase (AK) plays a crucial role in the survival of Daphnia magna, a water flea and a common planktonic invertebrate sensitive to water pollution, owing to the production of bioenergy. AK from D. magna (DmAK) has four highly conserved histidine residues, namely, H90, H227, H284, and H315 in the amino acid sequence. In contrast to DmAK WT (wild type), the enzyme activity of the H227A mutant decreases by 18%. To identify the structure-function relationship of this H227A mutant enzyme, the crystal 3D X-ray structure has been determined and an unfolding assay using anilino-1-naphthalenesulfonic acid (ANS) fluorescence has been undertaken. The results revealed that when compared to the DmAK WT, the hydrogen bonding between H227 and A135 was broken in the H227A crystal structure. This suggests that H227 residue, closed to the arginine binding site, plays an important role in maintaining the structural stability and maximizing the enzyme activity through hydrogen bonding with the backbone oxygen of A135.     

The crystal structures of 4-methoxybenzoate bound CYP199A2 and CYP199A4: structural changes on substrate binding and the identification of an anion binding site

The crystal structures of the 4-methoxybenzoate bound forms of cytochrome P450 enzymes CYP199A2 and CYP199A4 from the Rhodopseudomonas palustris strains CGA009 and HaA2 have been solved. The structures of these two enzymes, which share 86% sequence identity, are very similar though some differences are found on the proximal surface. In these structures the enzymes have a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. In both structures the substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of these enzymes for oxidative demethylation of the substrate. Mutagenesis studies on CYP199A4 highlight the involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates by these enzymes.     

Synthesis and structure of lower rim C-linked N-tosyl peptidocalix[4]arenes

Chiral p-tert-butylcalix[4]arenes functionalized at the lower rim with amino acid residues have been prepared. The (1)H and (13)C NMR spectra indicate that the macrocycles preferably adopt a cone conformation. Calix[4]arenes bearing amino acid moieties were prepared as a class of receptors selective for anions that are bound through hydrogen bonding with the NH group. The association constants are dependent on the nature of the substituents at the lower rim. Derivative 9 shows the strongest complexation and the largest selectivity for N-tosyl-(L)-alaninate. Finally, a preliminary X-ray crystal study of the difunctionalized receptor 6f shows the "flattened cone" conformation in the solid state.     

Biosynthetic phage display: a novel protein engineering tool combining chemical and genetic diversity

BACKGROUND: Molecular diversity in nature is developed through a combination of genetic and chemical elements. We have developed a method that permits selective manipulation of both these elements in one protein engineering tool. It combines the ability to introduce non-natural amino acids into a protein using native chemical ligation with exhaustive targeted mutagenesis of the protein via phage-display mutagenesis. RESULTS: A fully functional biosynthetic version of the protease inhibitor eglin c was constructed. The amino-terminal fragment (residues 8-40) was chemically synthesized with a non-natural amino acid at position 25. The remaining carboxy-terminal fragment was expressed as a 30-residue peptide extension of gIIIp or gVIIIp on filamentous phage in a phage-display mutagenesis format. Native chemical ligation was used to couple the two fragments and produced a protein that refolded to its active form. To facilitate the packing of the introduced non-natural amino acid, residues 52 and 54 in the carboxy-terminal fragment were fully randomized by phage-display mutagenesis. Although the majority of the observed solutions for residues 52 and 54 were hydrophobic - complementing the stereochemistry of the introduced non-natural amino acid - a significant number of residues (unexpected because of stereochemical and charge criteria) were observed in these positions. CONCLUSIONS: Peptide synthesis and phage-display mutagenesis can be combined to produce a very powerful protein engineering tool. The physical properties of the environment surrounding the introduced non-natural residue can be selected for by evaluating all possible combinations of amino acid types at a targeted set of sites using phage-display mutagenesis.     

竹红菌甲素与血红蛋白相互作用光谱

竹红菌甲素与血红蛋白相互作用光谱;马心血红蛋白;竹红菌甲素;相互作用;UV-Vis吸收光谱;荧光光谱;荧光猝灭动力学常数