山奈酚与水的氢键作用对其抗氧化活性影响的理论研究

本文用密度泛函理论在(RO)B3LYP/6-31G(2d,2p)水平上对山奈酚及其与水分子之间形成的氢键复合物进行结构优化,通过热力学计算研究了不同位置的酚羟基发生抽氢反应的键离解能(BDE)、质子解离反应过程的质子解离能(DPE)受分子间氢键的影响。结果表明:与H2O形成的分子间氢键会影响化合物结构,改变化合物B环与AC环的二面角,A5位酚羟基更容易发生抽氢反应和质子解离反应,此位点的BDE和DPE均明显降低,同时也降低C3位质子解离的DPE。分子间氢键的形成促使酚羟基的抽氢和质子解离反应,提高化合物抗氧化活性。     

去七肽胰岛素的分子动力学研究

本文用分子动力学的方法对去七肽胰岛素(DHPI)分子的构象进行了研究,首先用分子动力学方法对晶体胰岛素分子的构象能进行了优化,然后除去B链C端的最后七个残基(B24—B30),做分子动力学模拟,得到了DHPI的平衡构象和均方差波动。胰岛素分子的X射线晶体衍射结构和能量优化构象之间的均方根偏差为0.1;所得DHPI构象和胰岛素能量优化构象间C原子间的均方根偏差为1.8。变化最大的区域是A8—A10,A18—A21,B1—B41和B18—B23。     

虎纹捕鸟蛛毒素HWTX—Ⅱ的^1H—NMR信号归属和二级结构分析

虎纹捕鸟蛛毒素HWTX-Ⅱ是从虎纹捕鸟蛛Selenocosmia huwena的毒液中分离出的一种新型杀虫肽。应用2D－NMR技术研究该毒素分子的溶液结构特点,通过分析水及重水DQF－COSY、COSY、TOCSY和NOESY等^1H－NMR谱,识别出HWTX-Ⅱ全部37个氨基酸残基自旋体系;通过NOESY谱中的dαN、dαδ、dβN和dNN联系完成了序列专一的谱峰归属,确认了所有主链质子和除了Lys侧链εNH2质子外的所有侧链质子的化学位移,为完全解析HWTX-Ⅱ的溶液三维象奠定了基础,并且通过核磁数据分析,确定HWTX-Ⅱ的二级结构特点是含有较多的伸展构象,尤其是C端有一个典型的双股反平行的β折叠（Trp27-Cys29和Cys34-Lys36),分子中缺乏螺旋结构,这些二级结构特点与已探明结构的其他蜘蛛毒素的基本相同。     

胰岛素分子中的非螺旋氢键

以差值傅里叶技术对同晶置换途径测定的胰岛素结构进行精化后,用蛋白质分子内环境分析程序考查了胰岛素分子的非螺旋氢键。结果表明,几乎所有非螺旋氢键都集中分布在B链N端(B4—B6)和C端(B20—B26)伸展肽段,A链两段螺旋间的4肽(A9—A12)和C端5肽(A17—A21),及其之间。它们包括,A链和B链自身形成的氢键3对,A链与B链之间的氢键5对,侧链与主链间的氢键5对,以及分子间的氢键4对。它们对于胰岛素分子精细构象的形成和稳定,特别对于B链C端(B20—B26)肽段的特征折迭和A—B链的正确三维相关,具有重要作用。运用非螺旋氢键分析的结果,讨论了它们在蛋白质结构中可能的一般性作用,以及胰岛素C端和N端一系列修饰物结构-功能研究的结果,提出了胰岛素分子的二聚化及胰岛素原的C肽可能对分子的特征三维折迭具有保护性作用的看法。     

胰岛素分子中的非螺旋氢键

以差值傅里叶技术对同晶置换途径测定的胰岛素结构进行精化后,用蛋白质分子内环境分析程序考查了胰岛素分子的非螺旋氢键。结果表明,几乎所有非螺旋氢键都集中分布在B链N端(B4～B6)和C端(B20—B26)伸展肽段,A链两段螺旋间的4肽(A9—A12)和C端5肽(A17—A21),及其之间。它们包括,A链和B链自身形成的氢键3对,A链与B链之间的氢键5对,侧链与主链间的氢键5对,以及分子间的氢键4对。它们对于胰岛素分子精细构象的形成和稳定,特别对于B链C端(B20—B26)肽段的特征折迭和A—B链的正确三维相关,具有重要作用。运用非螺旋氢键分析的结果,讨论了它们在蛋白质结构中可能的一般性作用,以及胰岛素C端和N端一系列修饰物结构-功能研究的结果,提出了胰岛素分子的二聚化及胰岛素原的C肽可能对分子的特征三维折迭具有保护性作用的看法。     

变形菌视紫红质十年的回顾与展望

变形菌视紫红质(proteorhodopsin,PR)是一类吸光色素膜蛋白,它是由7个跨膜ɑ-螺旋(A～G)组成的视蛋白(opsins)与色素视黄醛(retinal)通过共价键结合而形成。PR广泛存在于海洋和淡水水域的微生物中,是一种质子泵型的视紫红质,可在光的驱动下将质子从细胞质泵到胞质间隙,从而在细胞内外产生质子梯度,形成的化学势能被用于合成ATP、物质的跨膜运输以及驱动鞭毛运动等方面。据估算含PR的细菌在海洋水域中约占总细菌的13%,而每个含PR的细胞中的PR分子数约为2.5×104个。对PR功能的研究表明PR具有增强其宿主菌抵抗外界不良环境的能力;而对于PR三维结构的研究对PR的作用机理及其功能的研究将有更进一步的推动作用。     

胰岛素B链N端和C端缩短对其与胰岛素抗体结合力的影响

本文研究了B链N端和C端缩短的若干胰岛素类似物与胰岛素抗体的结合能力。结果表明:去B链C端五肽胰岛素(DPI)分子中B_1-Phe去除后其与胰岛素抗体结合力明显下降,这与去B_1-Phe胰岛素与胰岛素抗体结合力下降的趋势相似;去B链C端六肽胰岛素(DHI)与胰岛素抗体结合力与DPI非常接近,都为胰岛素的70%左右。而去B链C端七肽胰岛素(DHPI)与胰岛素抗体结合力与去B链C端六肽胰岛素(DHI)相比,其结合力下降了一个数量级。说明胰岛素B_1-和B_(24)-Phe残基对组成和维持胰岛素分子的抗原决定簇起着重要作用。去B链九肽胰岛素(DNI)与胰岛素抗体的结合力与去B链C端八肽胰岛索(DOI)及DHPI相似。本文对上述结果进行了讨论。     

纳米狭缝中Aβ蛋白构象变化的间距作用

目的 金属表面对蛋白质分子具有吸附作用,然而在纳米尺度内,蛋白质分子构象受到狭缝的间距作用尚未明确。本文通过在分子动力学模拟中建立不同间距的金原子层,研究纳米级金属狭缝中蛋白质分子构象变化。方法 使用GOIPCHARMM力场在Au (111)金原子界面间对Aβ1-42蛋白单体进行分子动力学仿真,研究无狭缝的水溶液环境下和由5.0、5.5以及8.5 nm狭缝结构与Aβ蛋白相互作用及蛋白质构象变化。结果 当金狭缝结构间距从5.0 nm增加到8.5 nm,Aβ蛋白分子与两侧金层相互作用可由单表面吸附、双表面吸附过渡到无吸附。结论 Aβ蛋白分子在金狭缝结构中与表面发生相互作用,随狭缝间距和蛋白质分子距界面距离的变化,蛋白质分子的状态可能表现为单表面吸附、双表面吸附和无吸附3种状态。     

Val22对恶臭假单胞菌扁桃酸消旋酶催化活性的影响

恶臭假单胞菌扁桃酸消旋酶的Val22位于20 s环状结构上, 是与底物结合相关的氨基酸之一。其中Val被替换为Arg后酶活性下降了75.9%。除了酶与底物疏水作用减弱以外, 静电排斥作用增强也可能引起活性的下降。利用分子动力学模拟对酶与底物的米氏复合物进行分析, 结果表明: 突变后第22位氨基酸侧链与底物的静电势从0.036 kJ/mol升高至0.124 kJ/mol。这说明氨基酸侧链极性的改变增加了侧链与底物分子之间的静电排斥作用, 因而静电排斥作用也是导致突变体活性下降的原因之一。同时, 突变后系统势能增加了283 kJ/mol, 进一步证实了第22位氨基酸侧链极性和带电性质的改变导致酶与底物结合状态的势能增大, 从而引起活性大幅下降。因此, 将来对酶的结合口袋区域进行理性设计时, 除了考虑空间位阻效应外, 还需考虑疏水作用和静电作用。     

耐热甜味蛋白brazzein的溶液NMR结构研究——二级结构和分子骨架

brazzein是从非洲西部野生植物PentadiplandrabrazzeanaBaillon的果实中提取的一种甜味蛋白 .在所有已知的甜味蛋白质中 ,brazzein的分子量最小 ,水溶性最好 ,并且具有很好的热稳定性 .利用二维核磁共振 ( 2DNMR)技术研究brazzein的溶液三维结构 ,完成了包括主链和侧链在内的所有质子共振峰的序列归属 .brazzein的二级结构包含一段α螺旋 ( 2 1～ 2 9) ,一段较短的 310 螺旋 ( 1 4～ 1 7)和两股反平行 β折叠 ( 34～ 39,44～ 5 0 ) ,分子N端可能形成了第 3股 β链 ( 5～ 7) .比较研究发现 ,该甜味分子骨架CSαβ与蝎毒、昆虫防卫素和植物抗菌蛋白γ 硫素的分子支架基本相同 .以此为基础 ,讨论了这种多功能分子支架的意义 ,可能的甜味活性中心及其热稳定性的结构基础 .     

ZNC(C)PR及其类似物对大鼠脑内CNDF mRNA表达的影响

利用反转录ＰＣＲ的方法,以甘油醛３磷酸脱氢酶（ＧＡＰＤＨ）ｍＲＮＡ作为内源参照物,测定了记忆增强肽ＺＮＣ（Ｃ）ＰＲ及其类似物对大鼠海马和皮层中胆碱能神经分化因子（ＣＮＤＦ）ｍＲＮＡ表达的影响。ＺＮＣ（Ｃ）ＰＲ能显著地增强ＣＮＤＦｍＲＮＡ的表达,并在给药后１８ｈ达到高峰（海马３．０２倍,皮层５．３３倍,与对照组比较Ｐ＜０．０１）。ＺＮＣ（Ｃ）ＰＲ受体的激动剂ＮＬＰＲ也能诱导ＣＮＤＦｍＲＮＡ的表达,并比ＺＮＣ（Ｃ）ＰＲ活性更高。ＺＮＣ（Ｃ）ＰＲ受体的拮抗剂ＺＤＣ（Ｃ）ＰＲ能部分地阻断ＺＮＣ（Ｃ）ＰＲ的作用。精氨酸加压素（ＡＶＰ）只有微弱的活性,而催产素（ＯＸＴ）则没有活性。以上结果表明ＣＮＤＦ是ＺＮＣ（Ｃ）ＰＲ作用的靶基因之一,且这种作用是通过受体介导的。     

加压素片段类似物对C6细胞生长的影响

本文采用显微镜观察和逐个细胞突起长度测量及ＭＴＴ等方法研究了添加肽对无血清培养的Ｃ８细胞生长的影响：在培养早期（９－３６ｈ）,１０＾－８ｍｏｌ／Ｌ的ＡＶＰ、ＮＬＰＲ或ＺＮＣ（Ｃ）ＰＲ等都能刺激生长;同浓度的ＯＸＴ或ＺＤＣ（Ｃ）ＰＲ在起始时（９－１７ｈ）无明显影响,但稍后（３６ｈ）显示抑制作用。ＭＴＴ染色法分析的结果指出：神经肽促生长作用主要表现在细胞生长前期（１７ｈ）并且是肽浓度依赖的,ＺＮＣ（Ｃ     

ZNC(C)PR affects developmental changes of P46 phosphorylation in rat hippocampus

Protein phosphorylation has been suggested to be correlated with brain development and with the molecular mechanism of behavioral effects of neuropeptides. The present study reports in vitro endogenous phosphorylation of P46, a membrane-associated protein that is changed during development of the rat hippocampus. This study indicated that the degree of endogenous phosphorylation may be correlated with the establishment of synaptic connections. Interestingly, P46 was proved to be identical to a well-known growth-associated protein B-50/GAP-43 in its identical apparent molecular weight, isoelectric point, phosphorylation dependence, and the cross immunoreaction of monoclonal anti-B-50/GAP-43 antibody and P46. Moreover, neonatal administration of neuropeptide ZNC(G)PR could facilitate the developmental progress of P46 endogenous phosphorylation. It is suggested that the changes in P46 phosphorylation could be involved in the cellular mechanism of ZNC(C)PR behavioral effects on learning. © 1993 Wiley-Liss, Inc.     

Complete crystal structure of monocyte chemotactic protein-2, a CC chemokine that interacts with multiple receptors

Monocyte chemotactic protein 2 (MCP-2) is a CC chemokine that utilizes multiple cellular receptors to attract and activate human leukocytes. MCP-2 is a potent inhibitor of HIV-1 by virtue of its high-affinity binding to the receptor CCR5, one of the major coreceptors for HIV-1. Although a few structures of CC chemokines have been reported, none of these was determined with the N-terminal pyroglutamic acid residue (pGlu1) and a complete C-terminus. pGlu1 is essential for the chemotactic activity of MCP-2. Recombinant MCP-2 has Gln1 at the N terminus, 12-15% of which cyclizes automatically and forms pGlu1. The chemotactic activity of such MCP-2 mixture, which contains 12-15% pGlu1-form and 85-88% Gln1-form protein, is approximately 10 times lower when compared with that of fully cyclized MCP-2 preparation. Therefore, this chemokine is practically inactive without pGlu1. We have determined the complete crystal structure of MCP-2 that contains both pGlu1 and an intact C-terminus. With the existence of pGlu1, the conformation of the N-terminus allows two additional interactions between the two subunits of MCP-2 dimer: a hydrogen bond between pGlu1 and Asn17 and a salt bridge between Asp3 and Arg18. Consequently, both pGlu1 are anchored and buried, and thereby, both N-terminal regions are protected against protease degradation. We have also observed not previously reported extended helical nature of the C terminal region, which covers residues 58-74.     

Multi-drug resistance profile of PR20 HIV-1 protease is attributed to distorted conformational and drug binding landscape: molecular dynamics insights

The PR20 HIV-1 protease, a variant with 20 mutations, exhibits high levels of multi-drug resistance; however, to date, there has been no report detailing the impact of these 20 mutations on the conformational and drug binding landscape at a molecular level. In this report, we demonstrate the first account of a comprehensive study designed to elaborate on the impact of these mutations on the dynamic features as well as drug binding and resistance profile, using extensive molecular dynamics analyses. Comparative MD simulations for the wild-type and PR20 HIV proteases, starting from bound and unbound conformations in each case, were performed. Results showed that the apo conformation of the PR20 variant of the HIV protease displayed a tendency to remain in the open conformation for a longer period of time when compared to the wild type. This led to a phenomena in which the inhibitor seated at the active site of PR20 tends to diffuse away from the binding site leading to a significant change in inhibitor–protein association. Calculating the per-residue fluctuation (RMSF) and radius of gyration, further validated these findings. MM/GBSA showed that the occurrence of 20 mutations led to a drop in the calculated binding free energies (ΔG_bind) by ~25.17 kcal/mol and ~5 kcal/mol for p2-NC, a natural peptide substrate, and darunavir, respectively, when compared to wild type. Furthermore, the residue interaction network showed a diminished inter-residue hydrogen bond network and changes in inter-residue connections as a result of these mutations. The increased conformational flexibility in PR20 as a result of loss of intra- and inter-molecular hydrogen bond interactions and other prominent binding forces led to a loss of protease grip on ligand. It is interesting to note that the difference in conformational flexibility between PR20 and WT conformations was much higher in the case of substrate-bound conformation as compared to DRV. Thus, developing analogues of DRV by retaining its key pharmacophore features will be the way forward in the search for novel protease inhibitors against multi-drug resistant strains.     

Conformation and dynamics of drug-DNA intercalation.

Molecular dynamics simulations have been undertaken for a B-form dodecanucleotide duplex in solution with and without an intercalated proflavine molecule between the central C.G base pairs. The introduction of this simple intercalator affects both the conformational features and dynamic properties of the oligonucleotide double helix. Changes are seen in the rms atomic fluctuations and anisotropy of phosphate, sugar and base atoms. The backbone conformation is slightly changed on average and more sugars adopt the C3' endo conformation in the simulation of the complex compared with the simulation of the oligonucleotide alone. Both major and minor grooves becomes wider on average with the addition of the intercalating drug. Flanking A.T base pairs on both sides of the intercalation site have undergone an increase in flexibility, with the base pairs, especially at the 5' side, having the N1...N3 hydrogen bonds being broken.     

The peptide-receptive transition state of MHC class I molecules: insight from structure and molecular dynamics

MHC class I (MHC-I) proteins of the adaptive immune system require antigenic peptides for maintenance of mature conformation and immune function via specific recognition by MHC-I-restricted CD8(+) T lymphocytes. New MHC-I molecules in the endoplasmic reticulum are held by chaperones in a peptide-receptive (PR) transition state pending release by tightly binding peptides. In this study, we show, by crystallographic, docking, and molecular dynamics methods, dramatic movement of a hinged unit containing a conserved 3(10) helix that flips from an exposed "open" position in the PR transition state to a "closed" position with buried hydrophobic side chains in the peptide-loaded mature molecule. Crystallography of hinged unit residues 46-53 of murine H-2L(d) MHC-I H chain, complexed with mAb 64-3-7, demonstrates solvent exposure of these residues in the PR conformation. Docking and molecular dynamics predict how this segment moves to help form the A and B pockets crucial for the tight peptide binding needed for stability of the mature peptide-loaded conformation, chaperone dissociation, and Ag presentation.     

1H-NMR studies of receptor-selective substance P analogues reveal distinct predominant conformations in DMSO-d6

Proton nmr parameters are reported for DMSO-d6 solutions of two receptor-selective substance P analogues: Ac[Arg6,Pro9]SP6-11, which is selective for the NK-1 (SP-P) receptor and [pGlu6,N-MePhe8]SP6-11, which selectively activates the NK-3 (SP-N) receptor. Full peak assignments of both analogues were obtained by COSY experiments. The chemical shifts, coupling constants, and temperature coefficients of amide proton chemical shifts as well as NOESY effects and calculated side-chain rotamer populations of Phe side chains are reported for both peptides. Analysis of coupling constants and temperature coefficients together with the nuclear Overhauser enhancement spectroscopy effects suggest that Ac[Arg6,Pro9]SP6-11 has a trans configuration about the Phe8-Pro9 amide bond and the preferred conformation of this analogue has a type I beta-turn. The nmr data for [pGlu6,N-MePhe8]SP6-11 suggest that this peptide exists as a mixture of cis-trans isomers in which the cis isomer can preferably adopt a type VI beta-turn conformation, and the trans isomer can adopt a gamma-turn conformation. There are indications that the two last turns are stabilized by a hydrogen bond between the syn carboxamide proton and the pGlu ring carbonyl.     

Backbone 1H, 13C, and 15N chemical shift assignment for HIV-1 protease subtypes and multi-drug resistant variant MDR 769

HIV-1 protease (HIV-1PR) is an essential drug target in the treatment of patients infected with HIV-1. Mutations are found to arise in over 38 of 99 amino acid sites in this protein in response to drug therapy or natural selection, where many are found combinations that alter enzyme kinetics or inhibitor susceptibility without a clear structural mechanism. In efforts to understand how these mutations alter the flexibility and dynamics of HIV-1PR, we report the backbone ¹H, ¹³C, and ¹⁵N chemical shift assignments for subtypes C, circulating recombinant form CRF01_AE and a multi-drug resistant variant MDR 769. These assignments are essential for future work aimed at characterizing backbone dynamics, exchange dynamics and dynamics of protein/substrate or protein/inhibitor interactions.