固有无序超嗜热菌FlgM蛋白在两种不同温度下的构象变化特征

基于分子动力学模拟方法比较了超嗜热菌FlgM 蛋白在常温(293 K)和生理温度(358 K)下的结构特征.基于GROMACS软件包, 采用OPLS-AA分子力场和TIP3P水模型, 对超嗜热菌FlgM 蛋白在293 和358 K进行了2组独立的长时间分子动力学模拟, 每组体系模拟时间为1500 ns. 主要分析了两种温度下超嗜热菌FlgM蛋白的二级结构特征、整体构象变化及半无序化区域和结构化区域的构象特征. 研究结果表明: 在常温下, N端具有一定程度的螺旋成分, 但在生理温度下, 超嗜热菌FlgM 蛋白的结构变得松散, 螺旋结构减少, 构象稳定性减弱, H1 螺旋散开, FlgM 蛋白构象灵活性增强, 不稳定程度增加. 这些不同温度的结构变化说明: 半无序化区域(N端)在非结合状态下有一定的螺旋结构, 但该段螺旋的稳定性随温度升高而降低. 超嗜热菌FlgM蛋白会通过增加结构的无序程度使结构更加灵活, 以适应高温, 从而使该类固有无序蛋白更好地行使其功能, 如提高同其他成分的结合速率等.     

氯化癸铵的低频拉曼光谱研究

在290—340 K的温度范围内考察了氯化癸铵的低频拉曼光谱, 指认了210 cm~(-1)附近的谱带为分子烷基链的纵向声子振动带, 低于100 cm~(-1)的谱带为晶格振动带, 结果表明DeAC晶体存在313 K和321 K两个结构相变。在313 K以下的低温相, DeAC分子以完全有序的全反式链构象存在, 在313—321 K的中间相, 分子烷基链出现了旁式构象, 高温相与中间相相比主要是分子链横向堆积有序度明显降低, 而分子链构象特征无明显差异。     

四氯合锌酸二(正十一烷基铵)晶体相变的Raman光谱

用Raman光谱研究了[n-C_(11)H_(23)NH_3]_2ZnCl_4(简记为C_nZn)配合物的固-固相变.结果表明,配合物产生的固-固相变主要与烷烃链的堆积结构和分子构象变化有关.在 T_(c1)=25℃的相变是由于烷烃链的侧向堆积和分子构象的有序到部分无序变化.在中间相,分子链局部产生旁式构象.在T_(c2)=87℃的相变主要来源于烷烃链从部分构象有序到完全无序的变化.高温相形成了构象完全无序相.相应于烷烃链的“熔化”.     

分子对接和动力学模拟提高嗜热蛋白酶PhpI的活力

通过分子对接和动力学模拟对嗜热蛋白酶的分子进行改造, 确定蛋白酶PH1704(PhpI)定点突变残基, 并通过分子生物学实验进行验证. 突变体K43C的蛋白酶活力提高了5.8倍. 分子动力学模拟结果表明, 经过8 ns的动力学模拟后, K43C突变体二级结构由野生型的S2片层(F11-E12-D13)变成环状结构. E12和K43均是活性位点的重要残基, 这种变化将导致活性位点的柔性增强, 有利于催化反应的发生.     

温控分子动力学研究微管蛋白活性肽链的折叠机制

运用温控和常温分子动力学方法, 研究了微管蛋白活性部位Pep1-28肽链的折叠机制, 总模拟时间为380.0 ns. 对于温控分子动力学, 逐渐降温可以清晰显示Pep1-28肽链的折叠途径, 发生明显折叠的温度约为550 K, 其折叠和去折叠可逆机制为U(>1200 K)←→I1(1200-1000 K)←→I2(800 K)←→I3(600 K)←→I4(450 K)←→F1(400 K)←→F2(300 K), 其中U为去折叠态构象, I1、I2、I3和I4是折叠过程中的四个重要的中间态构象, F1和F2是两个结构相近的折叠态构象. 对于常温(300 K)分子动力学, 其构象转变和折叠过程相当迅速, 很难观察到有效、稳定的中间态构象. 尤其引人注意的是, 其折叠态结构陷入了能量局域极小点, 与温控(300 K)的有较大差异, 两者能量差高达297.53 kJ·mol-1. 可见, 温控分子动力学方法不仅清晰地显示多肽和蛋白质折叠过程的重要中间态构象, 为折叠和去折叠机制提供直接、可靠的依据, 而且还有助于跨越较高的构象能垒, 促使多肽和蛋白质折叠以形成全局能量最低的稳定结构.     

海藻糖抑制淀粉质多肽42构象转变的分子动力学模拟

应用分子动力学模拟方法研究了海藻糖抑制淀粉质多肽42(Aβ42)构象转变的分子机理.结果表明,海藻糖溶液浓度对Aβ42构象转变具有非常重要的影响.在水和低浓度海藻糖溶液(0.18mol·L-1)中,Aβ42可由初始的α-螺旋结构转变成β-折叠的二级结构;但海藻糖浓度为0.37mol·L-1时即可有效抑制Aβ42的构象转变.这是因为海藻糖利用其优先排阻作用使水分子在多肽周围0.2nm内富集,而其自身却在距离多肽0.4nm的位置附近团聚.另外,海藻糖还可通过降低多肽间的疏水相互作用,减少多肽分子内远距离的接触,有效抑制多肽的疏水塌缩和构象转变.上述分子模拟的结果对于进一步合理设计阿尔茨海默病的高效抑制剂具有非常重要的理论指导意义.     

振动光谱法研究水溶液中十六烷基三甲基溴化铵的分子构象

研究长链分子在水溶液中的分子构型有助于更好地理解生物膜的结构及生物大分子链(如蛋白链)的结构,受到人们的高度重视。许多表面活性剂分子的结构和性质已用各种物理和化学方法广泛地研究过,但用振动光谱方法(尤其是红外光谱法)研究水溶液中表面活性剂分子构象的报道则很少。十六烷基三甲基溴化铵(CTMAB)是一种典型的阳离子型表面活性剂,我们曾讨论了温度对CTMAB分子链亚晶胞堆积形式的影响。本文在280～320K范围     

蚕丝蛋白与类动物丝蛋白聚合物共混膜的振动光谱研究

蚕丝纤维具有优良的力学性能 ,不同的环境条件对其力学性能有一定的影响 ,但其力学性能主要取决于形成纤维过程中所形成的以分子链 β-折叠结构及其沿纤维轴方向高度取向为特征的丝纤维凝聚态结构 [1,2 ] .因此在丝纤维的形成及丝蛋白膜的人工制备过程中 ,丝蛋白分子链的构象及其构象转变一直是研究的重点[3～ 6 ] .以蚕丝蛋白 (Silk Fibroin,SF)稀溶液在常温下浇铸的 SF膜一般以无规线团 /α-螺旋为主的构象状态存在 ;经热处理、极性溶剂 (如甲醇等 )处理、应力作用或共混入一些能与SF形成分子间氢键的聚合物组分后 ,SF膜的构象将从无…     

羟丙基甲基纤维素诱导丝素蛋白的构象转变

制备了羟丙基甲基纤维素 (HPMC)和丝素蛋白 (SF)的共混膜 ,用FTIR ,XRD和DSC方法对共混膜的结构进行了表征 ,讨论了HPMC对SF的构象转变作用 ,结果表明 ,HPMC能够有效的诱导SF的构象转变 ,HPMC的比例是影响SF的构象转变程度的重要因素 .当混入 3%～ 10 %HPMC时 ,SF的构象存在由无规线团或SilkI向SilkII(β 折叠 )的转变 ,当加入 7%HPMC时 ,β 折叠构象的比例最大 .从红外分析可知 ,构象转变是由于适量的HPMC与SF混合形成了二者之间的分子间氢键所致 .对不同比例的共混膜测定其在水中的溶出率 ,结果显示当HPMC的比例为 7%时SF几乎不溶于水     

液/固界面多肽分子组装精细结构的扫描隧道显微镜研究

介绍了与蛋白构象病相关的淀粉样多肽分子组装结构的研究进展.综述了在固体、溶液以及界面等不同状态下多肽分子组装结构的表征方法,对于扫描隧道显微技术(STM)在解析多肽分子界面组装结构方面的研究进展进行了重点评述,主要包括在液/固界面上的多肽分子组装结构的精细特征,界面诱导的多肽构象转变,调节分子、染料等与多肽组装结构的相互作用模式和位点识别等.     

Mass spectrometric identification of an intramolecular disulfide bond in thermally inactivated triosephosphate isomerase from a thermophilic organism Methanocaldococcus jannaschii

The triosephosphate isomerase from the hyperthermophilic organism Methanocaldococcus jannaschii (MjTIM) is a tetrameric enzyme, with a monomer molecular mass of 23245 Da. The kinetic parameters, the k(cat) and the K(m) values, of the enzyme, examined at 25 °C and 50 °C, are 4.18 × 10(4) min(-1) and 3.26 × 10(5) min(-1) , and 0.33 and 0.86 mM(-1) min(-1) , respectively. Although the circular dichroism and fluorescence emission spectra of the protein remain unchanged up to 95 °C, suggesting that the secondary and tertiary structures are not lost even at this extreme temperature, surprisingly, incubation of this thermophilic enzyme at elevated temperature (65-85 °C) results in time-dependent inactivation, with almost complete loss of activity after 3 h at 75 °C. High-resolution electrospray ionization mass spectrometry (ESI-MS) reveals the monomeric mass of the heated sample to be 23243 Da. The 2 Da difference between native and heated samples suggests a probable formation of a disulfide bridge between proximal cysteine thiol groups. Liquid chromatography (LC)/ESI-MS/MS analysis of tryptic digests in the heated samples permits identification of a pentapeptide (DCGCK, residues 80-84) in which a disulfide bond formation between Cys81 and Cys83 was established through the collision-induced dissociation (CID) fragmentation of the intact disulfide-bonded molecule, yielding characteristic fragmentation patterns with key neutral losses. Neither residue is directly involved in the catalytic activity. Inspection of the three-dimensional structure suggests that subtle conformation effects transmitted through a network of hydrogen bonds to the active site residue Lys8 may be responsible for the loss of catalytic activity.     

Experiments with proteins at low temperature: what do we learn on properties in their functional state?

The authors compared the spectral response of Zn-substituted horseradish peroxidase in a glycerol/water solvent to hydrostatic pressure at 2 K and ambient temperature. The low temperature experiments clearly demonstrate the presence of at least three different conformations with drastically different elastic properties. However, the main conformation, which determines the fluorescence spectrum at ambient temperature, did not show any significant difference between low and high temperature and pressure. The authors conclude that the local compressibility of the heme pocket of the protein depends only very weakly on temperature.     

How Ligand Binding Affects the Dynamical Transition Temperature in Proteins

The biochemical functions of proteins are activated at the protein glass transition temperature, which has been proposed to be dependent upon protein-water interactions. However, at the molecular level it is unclear how ligand binding to well-defined binding sites can influence this transition temperature. We thus report molecular dynamics (MD) simulations of the ϵ subunit from thermophilic Bacillus PS3 in the ATP-free and ligand-bound states over a range of temperatures from 20 to 300 K, to study the influence of ligand association upon the transition temperature. We also measure the protein mean square displacement (MSD) in each state, which is well established as a means to quantify this dynamical temperature dependence. We find that the transition temperature is largely unaffected by ligand association, but the MSD beyond the transition temperature increases more rapidly in the ATP-free state. Our data suggests that ligands can effectively “shield” a binding site from solvent, and hence stabilize protein domains with increasing temperature.     

Hidden protein folding pathways in free-energy landscapes uncovered by network analysis

A network analysis is used to uncover hidden folding pathways in free-energy landscapes usually defined in terms of such arbitrary order parameters as root-mean-square deviation from the native structure, radius of gyration, etc. The analysis has been applied to molecular dynamics (MD) trajectories of the B-domain of staphylococcal protein A, generated with the coarse-grained united-residue (UNRES) force field in a broad range of temperatures (270K ≤ T ≤ 325K). Thousands of folding pathways have been identified at each temperature. Out of these many folding pathways, several most probable ones were selected for investigation of the conformational transitions during protein folding. Unlike other conformational space network (CSN) methods, a node in the CSN variant implemented in this work is defined according to the nativelikeness class of the structure, which defines the similarity of segments of the compared structures in terms of secondary-structure, contact-pattern, and local geometry, as well as the overall geometric similarity of the conformation under consideration to that of the reference (experimental) structure. Our previous findings, regarding the folding model and conformations found at the folding-transition temperature for protein A (Maisuradze et al., J. Am. Chem. Soc. 132, 9444, 2010), were confirmed by the conformational space network analysis. In the methodology and in the analysis of the results, the shortest path identified by using the shortest-path algorithm corresponds to the most probable folding pathway in the conformational space network.     

Magic-angle spinning solid-state NMR spectroscopy of the beta1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis

Magic-angle spinning solid-state NMR (SSNMR) studies of the beta1 immunoglobulin binding domain of protein G (GB1) are presented. Chemical shift correlation spectra at 11.7 T (500 MHz 1H frequency) were employed to identify signals specific to each amino acid residue type and to establish backbone connectivities. High sensitivity and resolution facilitated the detection and assignment of every 15N and 13C site, including the N-terminal (M1) 15NH3, the C-terminal (E56) 13C', and side-chain resonances from residues exhibiting fast-limit conformational exchange near room temperature. The assigned spectra lend novel insight into the structure and dynamics of microcrystalline GB1. Secondary isotropic chemical shifts report on conformation, enabling a detailed comparison of the microcrystalline state with the conformation of single crystals and the protein in solution; the consistency of backbone conformation in these three preparations is the best among proteins studied so far. Signal intensities and line widths vary as a function of amino acid position and temperature. High-resolution spectra are observed near room temperature (280 K) and at <180 K, whereas resolution and sensitivity greatly degrade substantially near 210 K; the magnitude of this effect is greatest among the side chains of residues at the intermolecular interface of the microcrystal lattice, which we attribute to intermediate-rate translational diffusion of solvent molecules near the glass transition. These features of GB1 will enable its use as an excellent model protein not only for SSNMR methods development but also for fundamental studies of protein thermodynamics in the solid state.     

A crystallographic, EPR and theoretical study of the Jahn-Teller distortion in [CuTp2] (Tp- = tris[pyrazol-1-yl]hydridoborate)

Crystals of the title compound (1) contain two independent, centrosymmetric half-molecules per asymmetric unit. While both of these show Jahn-Teller elongated six-coordinate geometries, the lengths of the elongated Cu-N bonds in the two molecules differ by 0.117(2) A at 30 K. The structure of one of these molecules (molecule A) does not vary with temperature below 350 K. The other molecule (molecule B) shows Cu-N bond lengths that are temperature-dependent between 225 and 375 K, but do not vary further at lower temperature. This indicates a fluxional axis of Jahn-Teller elongation in this molecule at these higher temperatures. Consideration of the thermal parameters in these structures implies that the fluxionality in molecule B is frozen out near 150 K. This conclusion is supported by a Q-band powder EPR study. The d-d transition energies of molecules A and B have been calculated by several density function (DF) methods, including a time-dependent DF calculation. The crystallographic data have been reproduced using the vibronic coupling model of Burgi and Hitchman. This has shown that the different fluxionality regimes for molecules A and B are not a consequence of their different static molecular structures, but rather reflect their different local environments in the crystal.     

Study on conformation interconversion of 3-alkyl-4-acetyl-3,4-dihydro-2H-1,4-benzoxazines from dynamic NMR experiments and ab initio density functional calculations

Variable-temperature NMR experiments and ab initio density functional calculations were carried out to investigate the conformation interconversion of novel chiral 3-alkyl-3,4-dihydro-2H-benzo[1,4]oxazine derivatives. With CDCl3 as the solvent, the coalescence temperatures of H2, H3, H11, and H19 of product 1 are about 289, 304, 292, and 316 K, with the corresponding activation free energies at 58.0 +/- 6.7, 60.9 +/- 7.1, 58.3 +/- 6.8, and 59.6 +/- 6.9 kJ.mol(-1), respectively. When dimethyl sulfoxide (DMSO-d6) was used as the solvent, 1H and 13C NMR signals were completely assigned at 375 K. The effects of solvent and temperature were investigated through a polarizable continuum model. At each theoretical level (MP2 or B3LYP), the changing tendencies of the calculated activation free energies and interconversion rates agree well with those of the NMR results. In addition, the interconversion rate at each specified temperature was calculated to be about 1.5 times faster in DMSO-d6 than in CDCl3. Accordingly, we failed to observe the coalescence phenomena of H3 and H19 in DMSO-d6 by NMR measurements from 296 to 375 K. The substitution effect at the R1-R5 positions was considered using density functional calculations, with the activation barriers decreasing as follows: product 6 > 3 > 1 > 7 > 2. This sequence is consistent with that of the reaction heats, except for product 7, implying that the interconversion processes may be thermodynamically controlled. Surprisingly, the substituted groups near the acetyl group in product 2 and 7 do not elevate the activation barrier but, instead, lower it somewhat, with the possible reasons for this provided in the paper.     

Effects of ionic strength, temperature and conformation on affinity interactions of β₂-glycoprotein I monitored by capillary electrophoresis

We have used CE to evaluate the interaction between β?-glycoprotein I (β?gpI) and heparin. β?gpI is a human plasma protein involved in the blood coagulation cascade. It is of interest to functionally characterize the interactions of β?gpI because the exact function is not entirely known and because circulating autoantibodies against β?gpI are associated with an increased risk of thrombotic events. The effect of the ionic strength, temperature, and conformation of the protein on the interaction between β?gpI and heparin has been studied. The CE procedure for this study is simple, fast, and automatic. β?gpI and heparin were allowed to interact during electrophoresis at different ionic strength buffers and at different capillary temperatures. To mimic perturbation of the conformation of β?gpI, different denaturing agents (SDS, ACN, and urea) were added to the BGE. While simple 1:1 binding isotherms were obtained at 22 °C, the data strongly suggest that at physiological temperature the binding stoichiometry is not 1:1 and/or that cooperative interactions begin to play a role. We found that (i) the K(D)-values differed by a factor of 60 at the ionic strengths studied (ii) β?gpI was resistant to denaturation with SDS and ACN, but was partially denatured by urea, and (iii) the K(D) for the β?gpI-heparin interaction in the presence of urea was ten times higher than the K(D) determined at the same conditions without urea added. Therefore, we conclude that the interaction between β?gpI and heparin is dependent on electrostatic interactions and on the conformation of β?gpI.