Contributions of a Position Amino Acid Residues to the Conformational Stability of GCN4 Leucine Zipper

Introduction Coiled coils[1] are widespread structures found in many natural proteins, such as structural proteins, transcrip- tion factors, receptor proteins, and enzymes[2,3]. Coils are the dominant structure in many fibrous proteins and the oligomeriza…     

Binding of vanadium compounds perturbs conformation and aggregation state of insulin

The interactions between zinc-free insulin and vanadium compounds, NaVO3, VO(acac)2 and VO(ma)2, have been investigated by fluorescence spectroscopy, circular dichroism (CD) and Fourier-transformed infrared (FT-IR) spectroscopy. The results showed that binding of vanadium compounds produced a static quenching of the intrinsic fluorescence of insulin. The apparent association constants were determined to be (0.17±0.01)×104 L*mol-1 for NaVO3, (2.8±0.2)×104 L*mol-1 for VO(acac)2, and (4.0±0.1)×104 L*mol-1 for VO(ma)2, respectively. The light scattering intensity of insulin decreased upon incubation with the vanadium compounds, suggesting the disaggregation of insulin. The attenuation of the band at 273 nm of insulin CD spectra also supported the disaggregation of insulin observed above. A new band at 1650～1653 cm-1 appeared in the FT-IR spectra of insulin upon incubation with the vanadium compounds, indicating the formation of an α-helix structure at B (9-19) motif. This α-helix structure suggests a structural change of insulin from an extended conformation (T state) to a helical conformation (R state), which is essential for binding of insulin to its receptor. In conclusion, binding of vanadium compounds results in conformational changes and disaggregation of insulin. These changes might account for the enhancement of binding affinity for insulin to its receptor in the presence of vanadium compounds.     

High-level expression and secondary structure analysis of the bovine mature prion protein

By using the recombinant DNA technology, the gene of the bovine mature prion protein (bPrPCL) has been cloned into pET30a and the resulting plasmid has been expressed in E.coli BL21(DE3). After solubilizing in 8 mol/L urea, the expression product was purified by cation ion exchange chromatography. The purified product was refolded by dilution and the recovery was about 15%. Analysis of mass spectrum, circular dichroism (CD) spectrum and Fourier transform infrared (FTIR) spectrum demonstrate that the molecular weight of the bPrPCL is 23 630 u, the bPrPCL has a high α-helix content (36.1%) and low β-sheet content (11.9%).     

Bis-methionine axial ligation of haem in bacterioferritin from Pseudomonas aeruginosa

The iron-containing bacterioferritins contain the protoporphyrin IX haem group. It has been established that Escherichia coli cytochrome b1, cytochrome b557 and bacterioferritin are identical. The optical spectra at room temperature of the haem group show it to be predominantly low-spin in both the ferrous and ferric states. The nature of the axial ligands binding the haem group to the polypeptide has, however, remained unknown. Low-spin, bis-coordinate haem centres in proteins typically have a role in rapid electron transfer as redox changes at the metal ion lead to little structural rearrangement. There are only four amino acids with side-chains that have ligand field strengths sufficient to generate the low-spin state of haem, namely, histidine, lysine, methionine and cysteine. Hence there are, potentially, ten different pairs of these four ligands which could be discovered in electron transfer haemoproteins. To date only three have been established with certainty. They are bis-histidine, as in mammalian cytochrome b5, methionine-histidine, typified by cytochrome c and lysine-histidine, recently recognized by spectroscopic methods in cytochrome f. Here we report the electron paramagnetic resonance and near infrared magnetic circular dichroism spectra of the oxidized state of Ps. aeruginosa bacterioferritin which enable the axial ligands to be identified as the thioether side chains of two methionine residues, a ligation scheme not previously reported for haem in any protein.     

富含羧基的多肽基双亲水杂化共聚物控制碳酸钙的形成

结合N-羧基-环内酸酐开环聚合和"巯-炔"光点击反应制备了侧链富含羧基的多肽基双亲水杂化共聚物(PEO-b-PPLG-g-MPA)。该多肽基共聚物可以模拟蛋白质指导CaCO3在水溶液中的形成。圆二色谱(CD)揭示了多肽链段在水溶液中的构象变化;扫描电子显微镜(SEM)揭示了矿化过程中CaCO3的形貌变化;X-射线衍射(XRD)确认了CaCO3的晶型。结果表明,该聚合物可以有效地控制CaCO3的形貌,此时多肽链段呈无规线团构象,而不是更为有序的α-螺旋或β-折叠构象;多肽链段的含量只有达到足够的浓度时,才能有效地控制的CaCO3晶体的生长;CaCO3的形貌则可以通过调节多肽链段的长度、溶液的pH值等因素进行调控,其可以是表面光滑的微球、表面堆满了CaCO3小颗粒的超结构微球、纳米棒或呈"花瓣状"聚集的纳米棒。XRD则证明形成的CaCO3呈稳定的方解石晶型。     

Spectroscopic studies on interaction of hemoglobin and serum albumin with nicotine

The interactions of nicotine and Hb/SA were studied in vitro by UV/Vis, fluorescence, 1H NMR and FT-IR spectroscopies. The UV/Vis absorbance of Hb/SA (200 nm)shifted to red and decreased gradually with the addition of nicotine, indicating that the protein conformational change resulted from the chemical interaction. With increasing nicotine concentration, incubation of SA with nicotine caused the quenching of fluorescence typical of protein tryptophan residues, which meant that the vicinity of the tryptophan residues of SA was changed because of nicotine. FT-IR spectra showed that α-helix component of Hb/SA decreased, turn and β-structure components of Hb/SA increased in the presence of nicotine. In the 1H NMR spectra of nicotine, all proton peaks on pyrrolidinyl ring moved to downfield and the resonance emanating from nicotine was preferentially broadened while the concentration of Hb/SA increased. All these results indicate that nicotine and Hb/SA in vitro interact on each other, forming a new complex and inducing the protein conformational change.     

邻苯二甲酸二丁酯与胰蛋白酶的相互作用

在模拟人体生理条件下(pH值为7.4),应用荧光光谱法、紫外光谱法和圆二色谱法(CD)并结合原子力显微镜(AFM)和分子模拟技术,研究了邻苯二甲酸二丁酯(DBP)对胰蛋白酶(Trypsin)的光谱性质、结构及催化活性的影响.结果表明,DBP通过氢键和范德华力与胰蛋白酶形成基态复合物而猝灭胰蛋白酶的内源荧光,DBP在胰蛋白酶上只有1个结合位点.同步荧光、紫外和CD光谱研究发现,DBP与胰蛋白酶的结合诱导了酶的α-螺旋、β-折叠和β-转角含量的减少,而增加了无规卷曲的含量.原子力显微镜图像显示,DBP的存在引起胰蛋白酶的表面形态发生变化,蛋白质发生了聚集.分子模拟结果表明,DBP结合于胰蛋白酶S1疏水空腔附近,与氨基酸His 57,Ser 195和Gly 193形成氢键.酶活测定结果显示,DBP的存在导致胰蛋白酶活性被抑制.     

A pentapeptide as minimal antigenic determinant for MHC class I-restricted T lymphocytes

Peptides that are antigenic for T lymphocytes are ligands for two receptors, the class I or II glycoproteins that are encoded by genes in the major histocompatibility complex, and the idiotypic alpha/beta chain T-cell antigen receptor. That a peptide must bind to an MHC molecule to interact with a T-cell antigen receptor is the molecular basis of the MHC restriction of antigen-recognition by T lymphocytes. In such a trimolecular interaction the amino-acid sequence of the peptide must specify the contact with both receptors: agretope residues bind to the MHC receptor and epitope residues bind to the T-cell antigen receptor. From a compilation of known antigenic peptides, two algorithms have been proposed to predict antigenic sites in proteins. One algorithm uses linear motifs in the sequence, whereas the other considers peptide conformation and predicts antigenicity for amphipathic alpha-helices. We report here that a systematic delimitation of an antigenic site precisely identifies a predicted pentapeptide motif as the minimal antigenic determinant presented by a class I MHC molecule and recognized by a cytolytic T lymphocyte clone.     

金属离子和变性剂对黄精凝集素Ⅱ构象与功能的影响

黄精凝集素II（Polygonatum cyrtonema Hua.Lectin II,PCLII)具有323nm的荧光发射峰和281．6nm的激发峰,其远紫外圆二色谱（CD谱）显示224nm负峰,分子中含有17．3％的α螺旋和45．8％的β折叠,在10mmol/L的SDS中,PCL II完全丧失兔红细胞凝集能力及促T－淋巴细胞有丝分裂活性,CD谱呈现双负峰的a螺旋构象,去除SDS后,则呈无规卷曲构象,此时,荧光发射峰红移而激发峰蓝移,PCL II仍完全失活,在EDTANa2溶液中,PCL II凝集红细胞能力及促淋巴细胞有丝分裂活性完全丧失,去除EDTA后,红细胞凝集活性完全恢复,但仍失去促淋巴细胞有丝分裂能力,PCL II二级结构变化不大,荧光光谱的变化表明微环境受到影响。     

大黄三种有效成分对胰蛋白酶活性及结构的影响

大黄素、大黄酸、大黄酚对胰蛋白酶活性均有一定程度的抑制作用.当有效成分浓度为2.5 mg·L~(-1)时,长濑法测定表明它们对胰蛋白酶的抑制率分别为38.0%、48.2%及35.4%.紫外吸收光谱、荧光光谱和圆二色谱研究表明三种有效成分影响胰蛋白酶酪氨酸、色氨酸和苯丙氨酸残基的微环境,改变了胰蛋白酶结构.圆二色谱计算发现,大黄素、大黄酸、大黄酚分别使胰蛋白酶二级结构中口α-螺旋含量减少了2.8%、3.2%和2.5%,β-折叠含量增加了1.6%、2.O%和1.4%.     

BRC2基元的合成及与靶肽p53(171-192)的相互作用

乳腺癌易感基因2(Breast Cancer Susceptibility Gene 2,BRCA2)是能抑制细胞发生恶变的关键基因.p53蛋白是参与细胞过程的主调控蛋白,p53蛋白要想行使其功能,则需要BRCA2及其产物对p53进行调控.选择BRCA2中的BRC2重复基元,用Fmoc固相合成法合成了BRC2及其3个片段.通过反相高效液相色谱(RPHPLC)和质谱对其进行表征,得到纯度大于90%的目标肽链.利用圆二色谱法初步探究了BRC2及其3个片段与靶肽p53(171-192)的相互作用.结果表明,BRC2的3个肽片段中,片段NEVGFRGFYSAHG在BRC2肽行使其功能中起着主要的作用.     

Electron transfer from protein to DNA in irradiated chromatin

Irradiation of dry or fully hydrated frozen DNA systems (conditions of direct damage) has been shown by electron-spin resonance spectroscopy to give rise to electron-gain centres localized on thymine (T.-) and electron loss centres ('holes') localized on guanine (G.+) with approximately equal yields. Our parallel studies on the development of both single- and double-strand breaks under comparable conditions provide good evidence that these radical centres are the precursors to such damage, and we and others have argued that this may be of relevance to the damage pathways in vivo. We now report evidence that when DNA is complexed to proteins as it is in the nuclei of eukaryotes, electron transfer from the histone to DNA is facile, leading to a significant increase in the yield of electron-gain centres in DNA as judged from their electron-spin resonance spectra. In contrast 'holes' generated in the protein are trapped and do not lead to any detectable increase in the yields of G.+.     

Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out in the middle.

We report here the determination and refinement to 1.9 A resolution by X-ray cryo-crystallography the structure of HLA-Aw68. The averaged image from the collection of bound, endogenous peptides clearly shows the atomic structure at the first three and last two amino acids in the peptides but no connected electron density in between. This suggests that bound peptides, held at both ends, take alternative pathways and could be of different lengths by bulging out in the middle. Peptides eluted from HLA-Aw68 include peptides of 9, 10 and 11 amino acids, a direct indication of the length heterogeneity of tightly bound peptides. Peptide sequencing shows relatively conserved 'anchor' residues at position 2 and the carboxy-terminal residue. Conserved binding sites for the peptide N and C termini at the ends of the class I major histocompatibility complex binding groove are apparently dominant in producing the long half-lives of peptide binding and the peptide-dependent stabilization of the class I molecule's structure.     

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.     

两栖类动物皮肤分泌抗菌肽的研究进展(动物科学)

近年因病原性细菌耐药性日趋严重,迫切需要从各种自然资源中寻找新的抗菌药物;因两栖类抗菌肽具有分子量小、水溶性好、抗原性低、抗菌作用强等优点,成为筛选抗菌药物的重要研究对象。本文就两栖抗菌肽的生物活性、分子结构、家族分类、抗菌分子机制、应用价值及研究前景等方面进行了综述。当前的研究表明,两栖类抗菌肽除具有抗菌作用外还有抗病毒抗肿瘤溶血等作用;它们因结构可分为3类:线性α-螺旋多肽、环性肽和含10～13个氨基酸残基长度的多肽;目前已发现抗菌肽有很多家族如Magainin家族、Aurein家族、Bombinin家族、Bombinin-like家族Brevinin家族等抗菌肽家族不同其生物活性也不同;它们杀菌主要通过膜裂解或膜不裂解机制完成;已发现两栖类抗菌肽在临床医药、食品保鲜防腐、农业生产等领域具有广阔的应用价值。随着在分子水平上深入的研究,两栖类抗菌肽将突破表达量低、特异性差、产量小、成本高等局限性,为人类制备新型抗菌药提供更多可用的资源。     

空间立体光滑倒角切削方法

提出了采用双链表翼边结构表示三维形体的几何信息和拓扑信息，运用ｄｏｏ－Ｓａｂｉｎ的双二次Ｂｅｚｉｅｒ曲面递归生成法及圆弧倒角法，实现空间立体的光滑倒角切削，并给出了运用该方法得到的实例．     

Circular dichroism spectral studies on the recombinant human neuroglobin

Neuroglobin (NGB) is a newly discov- ered member of the hemoglobin superfamily that is primarily expressed in the brain of humans and other vertebrates. The effects of protein concentration, solvent, pH and temperature on the secondary structure of NGB were investigated by employing far UV circular dichroism (CD) spectroscopy. The results show that NGB exists mainly in α-helix form when its concentration is less than 10 μmol/L. However, its α-helix content decreases with the increase of con- centration in the range of 10―40 μmol/L and remains unchanged when the concentration is higher than 40 μmol/L, which suggest that NGBs form intermolecular disulfide bond and aggregate in higher concentration. The α-helix content of NGB in methanol and ethanol is a little higher than that in water, indicating a higher stability of NGB in these solvents. NGB loses its α-helical secondary structure in either acidic or alka- line solution to some extent. Although increased temperature destabilizes the α-helices of NGB, over 16% of α-helices can be kept at 110°C. Therefore, NGB is a protein with hyperthermal stability.     

Identification of the T-cell and Ia contact residues of a T-cell antigenic epitope

The precise molecular structure of the antigenic determinant recognized by the T-cell receptor of the CD4-positive cell has not been completely resolved. A major advance in our understanding of this issue has been made by our demonstration of a direct association between an immunogenic peptide and a purified Ia molecule. The most likely and economical hypothesis is that antigen binds directly to an Ia molecule creating the antigenic determinant and that this antigen-Ia complex is recognized by the T-cell receptor. We examined in detail a determinant of hen egg-white lysozyme (HEL) contained in the tryptic fragment HEL(46-61), recognized by T cells in H-2k strains of mice. This peptide binds with a Kd of approximately 3 microM to I-Ak molecules. We have already ascertained that (1) the 10-mer HEL(52-61) is the shortest stimulatory peptide; (2) the Leu56 residue, the only residue different from mouse lysozyme, is responsible for the immunogenicity; (3) the Leu56 and Tyr53 residues are critical for recognition by the T-cell receptor and (4) HEL(46-61) generates multiple determinants when it associated with the I-Ak molecule. If antigen and Ia interact, the antigen must have two features: it must bind to an Ia molecule and also interact with the T-cell receptor. The two sites do not appear to be laterally separable in this peptide and are therefore probably composed of distinct but interspersed amino-acid residues. We have now identified the three residues of HEL(52-61) that contact the T-cell receptor and three other residues that contact the I-Ak molecule. From modelling studies we also propose that HEL(52-61) assumes an alpha-helical conformation as it is bound to I-Ak and recognized by the T-cell receptor.     

Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3

The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore. The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP-A (ref. 2). A conserved motif of CenH3, the CATD, defined by loop 1 and helix 2 of the histone fold, is necessary and sufficient for specifying centromere functions of CenH3 (refs 3, 4). The structural basis of this specification is of particular interest. Yeast Scm3 and human HJURP are conserved non-histone proteins that interact physically with the (CenH3-H4)(2) heterotetramer and are required for the deposition of CenH3 at centromeres in vivo. Here we have elucidated the structural basis for recognition of budding yeast (Saccharomyces cerevisiae) CenH3 (called Cse4) by Scm3. We solved the structure of the Cse4-binding domain (CBD) of Scm3 in complex with Cse4 and H4 in a single chain model. An α-helix and an irregular loop at the conserved amino terminus and a shorter α-helix at the carboxy terminus of Scm3(CBD) wraps around the Cse4-H4 dimer. Four Cse4-specific residues in the N-terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3(CBD) induces major conformational changes and sterically occludes DNA-binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome.