人类唾液蛋白N端片段的构型（英文）

人类唾液蛋白Statherin是含有43残基的酸性磷酸化蛋白.此蛋白对磷酸钙具有较高的吸附性,已知其N端15残基的片段(SN-15)会吸附于磷灰石的表面.在该工作中,作者以液体核磁共振波谱学研究SN-15的分子结构.圆二色光谱显示SN-15在磷酸盐缓冲溶液中具α螺旋构型.根据高分辨核磁共振氢谱(COSY,TOCSY及NOESY)作者得到相关的NOE及J耦合数据,氢氘交换实验也进一步显示SN-15具α螺旋构型.     

枯草芽孢杆菌双精氨酸转运系统TatAy 蛋白的溶液结构

存在于细菌和植物叶绿体中的双精氨酸(Tat)蛋白质转运系统能将底物蛋白以折叠的状态进行跨膜转运．该系统中的单次跨膜膜蛋白TatA 通过自身寡聚形成转运
底物蛋白的通道．该文应用液体核磁共振方法解析了枯草芽孢杆菌TatAy 蛋白在十二烷基胆碱磷酸胶束中的结构,它是由一个跨膜螺旋(TMH)和一个两亲性螺旋(APH)构成的L 型结构．通过与已经报道的枯草芽孢杆菌TatAd 蛋白的结构比较,该文能够鉴定出参与维持L 型构象的重要氨基酸残基,并指出了TatA 蛋白家族中若干较为保守的结构特征．在此基础上,该文讨论了保守残基在TatA 通道形成过程中可能发挥的作用．     

枯草芽孢杆菌双精氨酸转运系统TatA_y蛋白的溶液结构（英文）

存在于细菌和植物叶绿体中的双精氨酸(Tat)蛋白质转运系统能将底物蛋白以折叠的状态进行跨膜转运.该系统中的单次跨膜膜蛋白Tat A通过自身寡聚形成转运底物蛋白的通道.该文应用液体核磁共振方法解析了枯草芽孢杆菌Tat Ay蛋白在十二烷基胆碱磷酸胶束中的结构,它是由一个跨膜螺旋(TMH)和一个两亲性螺旋(APH)构成的L型结构.通过与已经报道的枯草芽孢杆菌Tat Ad蛋白的结构比较,该文能够鉴定出参与维持L型构象的重要氨基酸残基,并指出了Tat A蛋白家族中若干较为保守的结构特征.在此基础上,该文讨论了保守残基在Tat A通道形成过程中可能发挥的作用.     

应用~(15)N-~1H残留偶极偶合验证刪除肝癌2(DLC2-SAM)不育-α-基序域的螺旋间的取向(英文)

刪除肝癌2(DLC2),一种经常发现在原发性肝癌过低表达的肿瘤抑制基因,编码一种含有不育-α-基序多域蛋白质(DLC2-SAM).以前SAM域蛋白(DLC2-SAM)核磁共振结构显示此蛋白是由独特的四螺旋束组成,与其它已知SAM域结构截然不同.在该研究中,作者运用了15N-1H残留偶极偶合(RDC)作为附加约束连同NOE和TA-LOS数据进一步优化了DLC2-SAM的结构.由此所得的结构与没有15N-1H残留偶极偶合约束比较显示改善了结构的质量并且有较低的Q值.螺旋的取向,尤其是螺旋4,被残留偶极偶合数据所验证.RDC-优化的人类DLC2-SAM的结构与小鼠的DLC2-SAM很相像.DLC家庭独特的SAM域结构表明该域可能还具有没被发现的新功能.     

α-天门冬氨酰二肽酶及其进化酶的FT-IR研究

我们用FT IR方法 ,研究了α-天门冬氨酰二肽酶及其进化酶的二级结构 ,定量估算了天然酶的各种二级结构含量 ,α 折叠结构为 2 9% ,α 螺旋含量为 3 3 %～ 3 4% ,这与园二色谱测量α 螺旋为 3 3 %的结果有很好的一致 ,剩余的残基被认为形成不同类型的转角和无规结构。在进化酶中 ,β 折叠结构仍为 2 9% ,而α 螺旋为 3 0 %～ 3 1 % ,其它结构为不同类型的转角和无规结构。     

核磁共振螺旋快速成像的新进展

核磁共振快速成像能在几十毫秒内获取数据，对运动器官作适时显示，并在功能成像的研究等方面具有常规成像不能替代的优点，是核磁共振成像的发展方向．螺旋快速成像对硬件的要求较低，近年来方法上的改善，已使其趋于实用．文章简要介绍了螺旋快速成像原理及网格重建算法．     

5-羟基-7,4′-二甲氧基二氢黄酮分子结构的理论计算研究

分别应用HartreeFock从头算方法和B3LYP密度泛函方法从黑沙蒿中分离得到的5羟基7,4′二甲氧基二氢黄酮分子的几何构型进行优化,并采用规范不变原子轨道GIAO法,进行核磁共振化学位移计算,得到其两种构型的结构参数和NMR化学位移值,并对理论计算值与实验值的误差进行了统计分析.由计算结果推测该化合物分子结构中C(2)上的H在β位,即C(2)的绝对构型为S型.     

Pb2+对α-淀粉酶活性的影响及其光谱学研究

在α 淀粉酶介质中加入Pb2 ,通过光谱学手段研究Pb2 对α 淀粉酶活性影响的作用机理。结果表明低浓度的Pb2 对酶有激活作用 ,高浓度则严重抑制酶活性。在高浓度下 ,Pb2 能完全竞争出α 淀粉酶中的Ca2 而结合到了α 淀粉酶上 ,其EXAFS的测试表明Pb2 与氨基酸残基上的羧基氧发生了配位 ,配位数为 2 ,Pb—O键长为 0 2 34nm。圆二色 (CD)谱测试表明 ,高浓度的Pb2 结合使α 淀粉酶的二级结构被破坏 ,α 螺旋含量、β 转角及无规则卷曲大量下降 ,β 折叠、二硫键含量大量增多 ,Pb2 的这种完全结合致使酶的构象改变 ,形成无效的酶 Pb2 底物复合物 ,因而使酶失去活性。     

柠檬黄与玉米醇溶蛋白的相互作用研究

为了对玉米醇溶蛋白的改性研究及柠檬黄色素的安全使用评估提供理论依据,借助于荧光光谱法、紫外光谱法、全内反射-傅里叶红外光谱(ATR-FTIR)自去卷积计算及核磁共振氢谱研究了人工合成色素柠檬黄对玉米醇溶蛋白构象的影响。结果表明,柠檬黄对玉米醇溶蛋白有明显的荧光猝灭作用,猝灭机理较复杂。柠檬黄可以与玉米醇溶蛋白结合,分子间主要以疏水作用力结合,利用Stern-Volmer方程和Van't Hoff公式计算获得结合比为1∶1,结合常数K_a值较大。通过FTIR的自去卷积计算分析,这种结合反应导致玉米蛋白二级结构中α螺旋结构、β折叠和β转角均发生显著改变。核磁共振氢谱分析表明由于玉米醇溶蛋白具有两亲性,当溶剂为二甲基亚砜时,混合溶液为低极性环境,化学位移略向高磁场移动;当溶剂为重水D_2O时,化学位移明显地向低磁场移动,而且这种作用与反应时间长短无关。进一步说明在极性和低极性的环境下柠檬黄都会引起玉米醇溶蛋白构象的改变。     

S-构型转化对卵白蛋白微观结构的影响

采用圆二色谱(CD)、X射线衍射(XRD)、ANS荧光探针和紫外光谱(UV)研究了S-构型转化对卵白蛋白微观结构的影响.结果显示,不同诱导时间处理的卵白蛋白二级结构的α-螺旋,β-折叠,β-转角和无规卷曲之间相互转化,α-螺旋略有减少,β-折叠相应增加,分子有序性提高;S-构型转化后卵白蛋白晶体结构增加,72 h处理后...     

Probing electronic structure of biomineralized hydroxyapatite inside nanoclay galleries

Hydroxyapatite, the most abundant mineral in the human body, is also an important component in design of biomaterials for bone tissue regeneration. Synthetic hydroxyapatite mineralized in the laboratory often does not exhibit the same biological and morphological properties of biogenic hydroxyapatite in human bone. A biomimetic hydroxyapatite structure is synthesized using biomineralization routes inside the clay galleries of montmorillonite clay. Amino acids are used to modify the clay galleries. These amino acids are used to mineralize hydroxyapatite. The molecular interactions between nanoclay, modifiers inside nanoclay (amino acids) and biomineralized hydroxyapatite result in unique morphology, structure and stoichiometry of the biomineralized hydroxyapatite. Prior studies have indicated that this biomineralized hydroxyapatite inside nanoclay galleries is an effective component of tissue engineering bone scaffolds that elicits an optimal biological response from human mesenchymal stem cells. Here, a detailed electron energy-loss spectroscopy (EELS) study is reported that elucidates the differences in hydroxyapatite, biomineralized hydroxyapatite and β-tricalcium phosphate (β-TCP). Comparison of EELS low-loss transitions and energy loss near-edge structure (ELNES) of P-L_2,3 edges for these three compounds is done to determine if there are differences in their electronic structures. These changes observed experimentally are compared with prior predictions and simulations using molecular dynamics studies. The simulations predict attractive and repulsive interactions between phosphate, modified MMT clay and aminovaleric acid (amino acid) molecules. Kramers-Kronig analysis is performed on the loss spectra obtained to yield the real and imaginary parts of the dielectric function of the apatites (ε₁ and ε ₂). We have also used the ε₂ spectra obtained to calculate the AC conductivity spectra for the apatites. This study represents a unique experimental probe into molecular interactions in complex biomineralized hydroxyapatite structures. The small changes observed in the energy loss spectra appear to play important biological roles in biomineralized hydroxyapatite such as the ability to differentiate human mesenchymal stem cells into osteoblasts without growth media.     

Solid-state NMR studies of proteins immobilized on inorganic surfaces

Solid state NMR is the primary tool for studying the quantitative, site-specific structure, orientation, and dynamics of biomineralization proteins under biologically relevant conditions. Two calcium phosphate proteins, statherin (43 amino acids) and leucine rich amelogenin protein (LRAP; 59 amino acids), have been studied in depth and have different dynamic properties and 2D- and 3D-structural features. These differences make it difficult to extract design principles used in nature for building materials with properties such as high strength, unusual morphologies, or uncommon phases. Consequently, design principles needed for developing synthetic materials controlled by proteins are not clear. Many biomineralization proteins are much larger than statherin and LRAP, necessitating the study of larger biomineralization proteins. More recent studies of the significantly larger full-length amelogenin (180 residues) represent a significant step forward to ultimately investigate the full diversity of biomineralization proteins. Interactions of amino acids, a silaffin derived peptide, and the model LK peptide with silica are also being studied, along with qualitative studies of the organic matrices interacting with calcium carbonate. Dipolar recoupling techniques have formed the core of the quantitative studies, yet the need for isolated spin pairs makes this approach costly and time intensive. The use of multi-dimensional techniques to study biomineralization proteins is becoming more common, methodology which, despite its challenges with these difficult-to-study proteins, will continue to drive future advancements in this area.     

Rapid classification of protein structure models using unassigned backbone RDCs and probability density profile analysis (PDPA)

A method of identifying the best structural model for a protein of unknown structure from a list of structural candidates using unassigned 15N1H residual dipolar coupling (RDC) data and probability density profile analysis (PDPA) is described. Ten candidate structures have been obtained for the structural genomics target protein PF2048.1 using ROBETTA. 15N1H residual dipolar couplings have been measured from NMR spectra of the protein in two alignment media and these data have been analyzed using PDPA to rank the models in terms of their ability to represent the actual structure. A number of advantages in using this method to characterize a protein structure become apparent. RDCs can easily and rapidly be acquired, and without the need for assignment, the cost and duration of data acquisition is greatly reduced. The approach is quite robust with respect to imprecise and missing data. In the case of PF2048.1, a 79 residue protein, only 58 and 55 of the total RDC data were observed. The method can accelerate structure determination at higher resolution using traditional NMR spectroscopy by providing a starting point for the addition of NOEs and other NMR structural data.     

31P nmr study of I–IV group polycrystalline phosphates

Polycrystalline phosphates of group I–IV elements have been studied using solid state ³¹P NMR. The relationship between the spectral parameters (the principal components of the chemical shielding tensor and obtained from MAS values of the isotropic shifts of ³¹P) and the environment of the phosphorus atoms in these compounds has been considered. The shape of the lines in stationary ³¹P NMR spectra reflects the near neighbour environment of a phosphorus atom and the degree of distortion of the PO₄ tetrahedron. The type of cation, as well as the presence of the water of crystallization in the structure affect considerably an isotropic shift of ³¹P in phosphates. The high sensitivity of the solid state ³¹P NMR to even slight variations in the structure of the phosphate anions is shown.     

Laser-assisted production of tricalcium phosphate nanoparticles from biological and synthetic hydroxyapatite in aqueous medium

Pulsed laser ablation technique has attracted great attention as a method for preparing nanoparticles. In this work, calcined fish bones and synthetic hydroxyapatite, have been used as target to be ablated in de-ionized water with a pulsed CO₂ laser to produce calcium phosphate nanoparticles. The obtained nanoparticles were amorphous and spherical in shape with a mean diameter of about 25 nm. The microanalyses revealed that nanoparticles obtained from the synthetic HA undergo transformation to tricalcium phosphate. While nanoparticles obtained from the biological hydroxyapatite mostly preserve the composition of precursor material.     

Rotational resonance in uniformly 13C-labeled solids: effects on high-resolution magic-angle spinning NMR spectra and applications in structural studies of biomolecular systems

We describe investigations of the effects of rotational resonance (R(2)) on solid state (13)C NMR spectra of uniformly (13)C-labeled samples obtained under magic-angle spinning (MAS), and of the utility of R(2) measurements as structural probes of peptides and proteins with multiple uniformly labeled residues. We report results for uniformly (13)C-labeled L-alanine and L-valine in polycrystalline form, and for amyloid fibrils formed by the 15-residue peptide A beta(11-25) with uniform labeling of a four-residue segment. The MAS NMR spectra reveal a novel J-decoupling effect at R(2) conditions that may be useful in spectral assignments for systems with sharp (13)C MAS NMR lines. Pronounced dependences of the apparent isotropic (13)C NMR chemical shifts on MAS frequency near R(2) conditions are also observed. We demonstrate the feasibility of quantitative (13)C-(13)C distance determinations in L-valine, and qualitative determinations of inter-residue (13)C-(13)C contacts in A beta(11-25) fibrils. Finally, we demonstrate a "relayed" R(2) technique that may be useful in structural measurements on systems with poorly resolved (13)C MAS NMR lines.     

Optimized 3D-NMR sampling for resonance assignment of partially unfolded proteins

Resonance assignment of NMR spectra of unstructured proteins is made difficult by severe overlap due to the lack of secondary structure. Fortunately, this drawback is partially counterbalanced by the narrow line-widths due to the internal flexibility. Alternate sampling schemes can be used to achieve better resolution in less experimental time. Deterministic schemes (such as radial sampling) suffer however from the presence of systematic artifacts. Random acquisition patterns can alleviate this problem by randomizing the artifacts. We show in this communication that quantitative well-resolved spectra can be obtained, provided that the data points are properly weighted before FT. These weights can be evaluated using the concept of Voronoi cells associated with the data points. The introduced artifacts do not affect the direct surrounding of the peaks and thus do not alter the amplitude and frequency of the signals. This procedure is illustrated on 60-residue viral protein, which lacks any persistent secondary structure and thus exhibits major signal overlap.     

Double-quantum NMR spectroscopy based on finite pulse RFDR

We demonstrate that the finite pulse RFDR sequence (J. Chem. Phys. 114 (2001) 8473) can be used effectively for ³¹P double-quantum NMR spectroscopy at a spinning frequency of 10 kHz. The ³¹P NMR data measured for hydroxyapatite and octacalcium phosphate show that sizable double-quantum excitation efficiency can be obtained with the ratio of the recoupling field to spinning frequency set equal to 1.67.     

A new approach to visualizing spectral density functions and deriving motional correlation time distributions: applications to an alpha-helix-forming peptide and to a well-folded protein

A new approach to visualizing spectral densities and analyzing NMR relaxation data has been developed. By plotting the spectral density function, J(omega), as F(omega)=2 omega J(omega) on the log-log scale, the distribution of motional correlation times can be easily visualized. F(omega) is calculated from experimental data using a multi-Lorentzian expansion that is insensitive to the number of Lorentzians used and allows contributions from overall tumbling and internal motions to be separated without explicitly determining values for correlation times and their weighting coefficients. To demonstrate the approach, (15)N and (13)C NMR relaxation data have been analyzed for backbone NH and C(alpha)H groups in an alpha-helix-forming peptide 17mer and in a well-folded 138-residue protein, and the functions F(omega) have been calculated and deconvoluted for contributions from overall tumbling and internal motions. Overall tumbling correlation time distribution maxima yield essentially the same overall correlation times obtained using the Lipari-Szabo model and other standard NMR relaxation data analyses. Internal motional correlational times for NH and C(alpha)H bond motions fall in the range from 100 ps to about 1 ns. Slower overall molecular tumbling leads to better separation of internal motional correlation time distributions from those of overall tumbling. The usefulness of the approach rests in its ability to visualize spectral densities and to define and separate frequency distributions for molecular motions.     

Investigation on the Beckmann rearrangement reaction catalyzed by porous solids: MAS NMR and theoretical calculations

In the last years, ‘in situ’ solid-state NMR has been applied to investigate the Beckmann rearrangement of oximes into amides using zeolites and mesoporous materials of different structure containing Brønsted acids or silanol groups as active sites. DFT methods have been applied to model the geometry of the complexes resulting from adsorption of reactants, reaction intermediates and products on clusters representing the zeolite centers, and their ¹⁵N and ¹³C NMR chemical shift calculated theoretically. This article reviews the results reported in the bibliography on the Beckmann rearrangement of various oximes (acetophenone oxime, cyclohexanone oxime and cyclododecanone oxime) mainly using ‘in situ’ ¹⁵N NMR spectroscopy and theoretical calculations, and are compared with those obtained by ‘in situ’ infrared spectroscopy. The combination of experiment and theory has been shown to be very useful for the interpretation of the NMR spectra and the identification of the species present at different reaction temperatures.