Optimal isotope labelling for NMR protein structure determinations

Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Here we present stereo-array isotope labelling (SAIL), a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination.     

Demonstration by genetic suppression of interaction of GroE products with many proteins

The way in which proteins attain and maintain their final form is of fundamental importance. Recent work has focused on the role of a set of ubiquitous proteins, termed chaperonins, in the assembly of phage and multisubunit proteins. The range of chaperonin action is unknown; they could interact with most cellular polypeptides or have a limited subset of protein partners. Included in the chaperonin family is the essential heat-shock regulated Escherichia coli groEL gene product. Over-expression of the groE operon in E. coli causes enhanced assembly of heterologously expressed ribulose bisphosphate carboxylase subunits and suppresses the heat-sensitive mutant phenotype of several dnaA alleles. It has been inferred that suppression of heat-sensitive mutations is confined to dnaA alleles and that this confinement could reflect an interaction between the groE operon products and a dnaA protein aggregate at the replication origin. We now report that multiple copies of the groE operon suppress mutations in genes encoding several diverse proteins. Our data indicate a general role for the groE operon products, the GroEL and GroES proteins, in the folding-assembly pathways of many proteins.     

Eukaryotic type II chaperonin CCT interacts with actin through specific subunits

Chaperonins assist the folding of other proteins. Type II chaperonins, such as chaperonin containing TCP-1(CCT), are found in archaea and in the eukaryotic cytosol. They are hexadecameric or nonadecameric oligomers composed of one to eight different polypeptides. Whereas type I chaperonins like GroEL are promiscuous, assisting in the folding of many other proteins, only a small number of proteins, mainly actin and tubulin, have been described as natural substrates of CCT. This specificity may be related to the divergence of the eight CCT subunits. Here we have obtained a three-dimensional reconstruction of the complex between CCT and alpha-actin by cryo-electron microscopy and image processing. This shows that alpha-actin interacts with the apical domains of either of two CCT subunits. Immunolabelling of CCT-substrate complexes with antibodies against two specific CCT subunits showed that actin binds to CCT using two specific and distinct interactions: the small domain of actin binds to CCTdelta and the large domain to CCTbeta or CCTepsilon (both in position 1,4 with respect to delta). These results indicate that the binding of actin to CCT is both subunit-specific and geometry-dependent. Thus, the substrate recognition mechanism of eukaryotic CCT may differ from that of prokaryotic GroEL.     

Quantitative dynamics and binding studies of the 20S proteasome by NMR

The machinery used by the cell to perform essential biological processes is made up of large molecular assemblies. One such complex, the proteasome, is the central molecular machine for removal of damaged and misfolded proteins from the cell. Here we show that for the 670-kilodalton 20S proteasome core particle it is possible to overcome the molecular weight limitations that have traditionally hampered quantitative nuclear magnetic resonance (NMR) spectroscopy studies of such large systems. This is achieved by using an isotope labelling scheme where isoleucine, leucine and valine methyls are protonated in an otherwise highly deuterated background in concert with experiments that preserve the lifetimes of the resulting NMR signals. The methodology has been applied to the 20S core particle to reveal functionally important motions and interactions by recording spectra on complexes with molecular weights of up to a megadalton. Our results establish that NMR spectroscopy can provide detailed insight into supra-molecular structures over an order of magnitude larger than those routinely studied using methodology that is generally applicable.     

三价金属铊与配体邻啡咯啉和8-羟基喹啉络合物的合成及结构表征

合成了新的三价金属铊与配体邻啡咯啉(Phen)和8-羟基喹啉(Oxine)的络合物,并应用多核核磁共振(1H,13C和205Tl)渡谱和振动光谱,系统地对所合成的络合物进行了溶液和固相的表征.204T1核磁共振波谱表明,单配体络合物[T1(phen)] 3+,双配体络合物[T1(phen)2] 3+和三配体络合物[T1(phen)3] 3+以化学平衡的形式存在于二甲基砜(DMSO)和乙腈(CH3CN)溶液中.并在DMSO溶液中,测定了三配体络舍物[T1(phen)3] (ClO4)3 (1)和四配体络舍物ETl(oxine)4] (ClO4)3(2)的1H和13C NMR谱,其自旋-自旋耦合常数(T1(I=1/2)-13C和Tl(I=1/2)-1H)均被观测到.从二维共振谱(13C-1H COSY图谱),四配体络合物FTl(oxine)4] 3+中C-H联结和其复杂的自旋-自旋耦合1H NMR图谱均被明确地归属和指认.紫外-可见光谱表明配体到金属的电荷转移吸收谱(LMCT)分别为315 nm(ε=1.65×103mol-1·cm-1)(1)和375 nm(ε=7.48×103mol-1·cm-1)(2).由此可见,三价铊与配体邻啡咯啉(Phen)和8-羟基喹啉(Oxine)均都生成了稳定的络合物.     

尾式酪氨酸卟啉及其金属配合物的合成

报道了５－［（对－Ｎ－酪氨酸丁氧基）苯基］－１０,１５,２０－三（对－氯苯基）卟啉（Ｈ２Ｌ）及其配合物ＭＬＣ１（Ｍ＝Ｃｏ,Ｆｅ,Ｍｎ）的合成,经元素分析、ＵＶ－ｃｉｓ、荧光光谱、ＩＲ和＾１ＨＮＭＲ等结构表征。催化实验发现除配体外,３种配合物在分子氧氧化苯甲醛的反应中具有催化作用。     

七元瓜环与吖啶类化合物主客体作用测试

利用紫外吸收光谱法、荧光光谱法和1H NMR技术等现代分析方法,测试了七元瓜环(cucurbit[7]urils)与吖啶、9-氨基吖啶、3,6-二氨基吖啶(分别记为g1、g2、g3)的主客体作用。实验结果表明:在酸性或中性条件,用三种方法均能观察到七元瓜环与客体的相互作用,但在强碱性条件下,用上述方法观察不到主客体的相互作用,利用光谱法得到七元瓜环与g1、g2、g3均形成物质的量之比为1∶1的包合物,计算出相应的平衡常数,并利用核磁表征考察了主客体可能的作用模式。     

氨基酸希夫碱及其金属锌配合物的合成及表征

 合成了5种氨基酸希夫碱Sal-Gly(甘氨酸希夫碱)、Sal-Glu(谷氨酸希夫碱)、Sal-Met(甲硫氨酸希夫碱)、Sal-Tyr(酪氨酸希夫碱)、Sal-Arg(精氨酸希夫碱)及其金属锌离子配合物共10种化合物,采用核磁共振、红外光谱、紫外可见光谱和荧光光谱进行表征。     

Different conformations for the same polypeptide bound to chaperones DnaK and GroEL.

The proteins DnaK (hsp70) and GroEL (cpn60) from Escherichia coli are prototypes of two classes of molecular chaperones conserved throughout evolution. The analysis of transferred nuclear Overhauser effects in two-dimensional NMR spectra is ideally suited to determine chaperone-bound conformations of peptides. The peptide vsv-C (amino-acid sequence KLIGVLSSLFRPK) stimulates the ATPase of BiP and Hsc70 (ref. 3) and the intrinsic ATPase of DnaK. The affinity of the vsv-C peptide for DnaK is greatly reduced in the presence of ATP. Here we analyse transferred nuclear Overhauser effects and show that the peptide is in an extended conformation while bound to DnaK but is helical when bound to GroEL. NMR also indicates that the mobility of the peptide backbone is reduced more by binding to DnaK than by binding to GroEL, whereas the side chains are less mobile when bound to GroEL.     

5—磺基水杨酸存在下两相合成二茂钛氨基酸配合物

在两相体系（H2O／CHCl3)中,利用二氯二茂钛与5－磺基水杨酸形成的水溶液和N－（取代苯基）氨基乙酸反应,简便全盛了三种二茂钛氨基酸配合物,采用元素分析、IR及^1H NMR等对其进行了表征。结果表明,氨基酸的羧基以单齿氧原子与钛配位,形成了双分子取代的二茂钛配合物。     

Study of complexes of lanthanum with amino acids by titration calorimeter

The stability constants and thermodynamic functions for complexes of lanthanum with eight kind of amino acids according to 1∶1 and 1∶2 in proportion have been determined by titration calorimeter at 298.15 K. The enthalpy change makes a predominant contribution to the stability of these complexes. The ring in amino acid associated with lanthanum ion helps to enhance the stability of complexes. Steric effects between rings in complexes leads to that the equilibrium constants of reaction of the complexes (1∶2) is much less than that of the complexes (1∶1). Foundation item: Supported by the National Nature Science Foundation of China (29873036) Biography: Ye Gang (1974-), male, M S., research direction: thermochemistry.     

2,5—噻吩二甲醛双异烟酰腙与某些二价3d过渡金属配合物的合成、表征及抗肿瘤活性

合成了了2,5—噻吩二甲醛双异烟酰腙(H_2TPI)及其与钴(Ⅱ)、镍(Ⅱ)和铜(Ⅱ)的配合物[M(TPI)·nH_2O,M=Co(Ⅱ)、Ni(Ⅱ)和Cu(Ⅱ),n=3,4]。通过元素分析、红外光谱、紫外可见光谱、磁矩、~1H核磁共振谱、ESR谱、差热分析和x—射线粉末衍射分析等,研究了配合物的组成和性质,并对配合物的抗肿瘤活性作了初步探索。     

Solution structure of the major binding protein for the immunosuppressant FK506

The major FK506 binding protein (FKBP, relative molecular mass approximately 11,800; Mr 11.8K) and cyclophilin (Mr approximately 17K) belong to a class of proteins termed immunophilins. Although unrelated at the amino-acid sequence level, they both possess peptidyl-prolyl cis-trans isomerase activities which are inhibited by immunosuppressants that block signal transduction pathways leading to T-lymphocyte activation. FK506 and rapamycin strongly inhibit the peptidyl-prolyl cis-trans isomerase activity of FKBP, whereas cyclosporin A inhibits that of cyclophilin. The significance of this enzyme activity and the role of the immunophilins in immunoregulation is unknown. To understand better the function of the immunophilins and their interaction with inhibitors, we are investigating the solution structures of FKBP and FKBP-inhibitor complexes by multidimensional NMR methods. Here we report the solution conformation of FKBP, as generated by NMR, distance geometry and molecular dynamics methods. The regular secondary structure of FKBP is composed mainly of beta sheet (approximately 35%) with little helical structure (less than 10%). The hydrophobic core of the molecule, containing the buried side chains of six of the protein's nine aromatic amino acids, is enclosed by a five-stranded antiparallel beta sheet on one side, a loop and a short helix at residues 51-56 and 57-65, and an aperiodic loop at residues 81-95. Examination of the structure suggests a possible site of interaction with FK506.     

新型1,8-萘啶氟硼化合物的合成、光物理性质及理论计算

以5,7-二甲基-2-氨基-1,8-萘啶为原料,通过2步不同的N-酰基化反应得到苯甲酰胺衍生物(化合物L1),进而与BF_3·Et_2O在三乙胺的存在下合成得到一个新型N,O-配位1,8-萘啶氟硼化合物(化合物T1),其结构经核磁共振谱(~1H NMR,~(19)F NMR)、质谱和红外光谱表征.对化合物T1的紫外-可见吸收和荧光发射光谱进行研究,表明化合物T1具有良好的光物理性质,如摩尔消光系数大、荧光量子产率高和固体发光.此外,其实验结果用密度泛函理论计算加以验证.     

丙烯氢甲酰化HRh（CO）（PPh3）3／SiO2催化剂的NMR研究

用固定床加压气相连续流动微反－气相色谱组合装置评价了ＳｉＯ２负载铑膦配合物催化剂上丙烯氢甲酰化反应．通过溶液（或液膜）３１Ｐ（１Ｈ）ＮＭＲ谱表征,发现配合物ＨＲｈ（ＣＯ）（ＰＰｈ３）３负载于硅胶载体上之后,原负载前驱苯溶液３１Ｐ（１Ｈ）ＮＭＲ谱在４１．１ｐｐｍ处的特征峰消失,但在化学位移为３７．０ｐｐｍ附近出现一新的宽峰,后者可能缘于配合物与载体表面发生相互作用．反应４ｈ后工作态催化剂的ＮＭＲ测试表明,在丙烯氢甲酰化反应条件下,铑的三膦配合物发生部分解络;工作态催化剂表面上主要的铑膦催化活性物种是含双膦的配合物物种,并暗示存在着含双羰基或羰、酰基的反应中间态;同时也观察到可归属于解络后的游离三苯基膦物种的相应谱峰．反应９０ｈ后催化剂的ＮＭＲ谱显示,铑配合物催化剂中膦配体含量已相当低;反应过程中膦配位体被氧化及铑、磷组份的流失是负载型催化剂活性下降乃至失活的主要原因．     

N,N-二甲基- N,N-二苯基-3,6-二氧杂辛二酰胺氯化稀土配合物的合成及表征

在非水介质中合成了六种轻稀土氯化物与配体 N,N′-二甲基 - N,N′-二苯基 - 3,6 -二氧杂辛二酰胺 (DMDD)形成的固体配合物 ,通式为 [L n(DMDD) Cl2 (H2 O) 2 ]Cl(Ln =L a～ Eu,不包括Pm) .用元素分析、摩尔电导、红外光谱、核磁共振和 TG- DTA分析对这些配合物进行了表征 .结果表明 ,在这些新配合物中 ,配体 DMDD表现为四齿配位行为     

腐殖酸与稀土元素的结合点位及其表征研究

采用红外光谱(FTIR)与核磁共振波谱(1H NMR和13C NMR)技术,对来自森林土壤和湖泊沉积物的两种腐殖酸及其与三价稀土离子(RE3 )沉淀反应的产物进行对比研究.结果表明,腐殖酸结构中活性官能团特征以及它的拥有量因腐殖化程度不同而存在较大差异.腐殖酸与RE3 沉淀反应产物在性质上属于一种有机弱酸盐,羧基-COOH和酚羟基-OH为主要结合点位,结合反应过程中它们的质子被RE3 交换出来.     

对─叔丁基─杯［8］芳烃对C_（60）的分子识别

通过甲苯溶液中对─叔丁基─杯［８］芳烃与Ｃ６０的反应，合成了它们的１：１络合物．产物经红外光谱、元素分析、Ｘ－射线粉末衍射确证．还采用固体１３Ｃ核磁共振技术研究对─叔丁基─杯［８］芳烃对Ｃ６０的分子识别．１３Ｃ魔角固体核磁共振谱清楚地揭示出Ｃ６０被包结在杯芳烃的疏水空腔中．１３Ｃ核磁共振静态谱表明被包在空腔中的Ｃ６０分子于室温时仍在快速旋转．这说明Ｃ６０分子与对─叔丁基─杯［８］芳烃之间的相互作用非常弱．     

石墨炉原子吸收法间接测定氨基酸注射液中游离氨基酸含量

采用Pd-Rh化学基体改进剂,建立了石墨炉原子吸收光谱法（GFAAS）间接测定游离氨基酸的新方法．本方法基于磷酸铜悬浮液与氨基酸发生络合反应生成铜-氨基酸络合物,铜络合物可以采用GFAAS进行测定,而后根据铜-氨基酸络合物中的铜与氨基酸的摩尔比,依据铜的浓度计算出氨基酸的浓度．实验考察了多种金属离子化合物与氨基酸的反应,最终选择了硫酸铜悬浮液．实验选择Pd-Rh化学基体改进剂做铜的稳定剂,热解温度可以达到1630℃．在最佳实验条件下,成功地测定了氨基酸注射液中游离氨基酸含量,回收率为92%～108%．