3D QSAR studies on peroxisome proliferator-activated receptor γ agonists using CoMFA and CoMSIA

The peroxisome proliferator-activated receptors (PPARs) have increasingly become attractive targets for developing novel anti-type 2 diabetic drugs. We employed comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) to study three-dimensional quantitative structure–activity relationship (3D QSAR) based on existing agonists of PPAR (including five thiazolidinediones and 74 tyrosine-based compounds). Predictive 3D QSAR models with conventional r² and cross-validated coefficient (q²) values up to 0.974 and 0.642 for CoMFA and 0.979 and 0.686 for COMSIA were established using the SYBYL package. These models were validated by a test set containing 18 compounds. The CoMFA and CoMSIA field distributions are in general agreement with the structural characteristics of the binding pockets of PPAR, which demonstrates that the 3D QSAR models built here are very useful in predicting activities of novel compounds for activating PPAR.      

Defining the minimum size of a hydrophobic cluster in two-stranded alpha-helical coiled-coils: effects on protein stability

The alpha-helical coiled-coil motif is characterized by a heptad repeat pattern (abcdefg)(n) in which residues a and d form the hydrophobic core. Long coiled-coils (e.g., tropomyosin, 284 residues per polypeptide chain) typically do not have a continuous hydrophobic core of stabilizing residues, but rather one that consists of alternating clusters of stabilizing and destabilizing residues. We have arbitrarily defined a cluster as a minimum of three consecutive stabilizing or destabilizing residues in the hydrophobic core. We report here on a series of two-stranded, disulfide-bridged parallel alpha-helical coiled-coils that contain a central cassette of three consecutive hydrophobic core positions (d, a, and d) with a destabilizing cluster of three consecutive Ala residues in the hydrophobic core on each side of the cassette. The effect of adding one to three stabilizing hydrophobes in these positions (Leu or Ile; denoted as [see text]) was investigated. Alanine residues (denoted as [see text]) are used to represent destabilizing residues. The peptide with three Ala residues in the d a d cassette positions ([see text]) was among the least stable coiled-coil (T(m) = 39.3 degrees C and Urea(1/2) = 1.9 M). Surprisingly, the addition of one stabilizing hydrophobe (Leu) to the cassette or two stabilizing hydrophobes (Leu), still interspersed by an Ala in the cassette ([see text]), also did not lead to any gain in stability. However, peptides with two adjacent hydrophobes in the cassette ([see text])([see text]) did show a gain in stability of 0.9 kcal/mole over the peptide with two interspersed hydrophobes ([see text]). Because the latter three peptides have the same inherent hydrophobicity, the juxtaposition of stabilizing hydrophobes leads to a synergistic effect, and thus a clustering effect. The addition of a third stabilizing hydrophobe to the cassette ([see text]) resulted in a further synergistic gain in stability of 1.7 kcal/mole (T(m) = 54.1 degrees C and Urea(1/2) = 3.3M). Therefore, the role of hydrophobicity in the hydrophobic core of coiled-coils is extremely context dependent and clustering is an important aspect of protein folding and stability.     

大鼠尿激酶SYBR Green I real time RT-PCR引物的设计

设计用于SYBR Green I法实时定量逆转录多聚酶链反应(QRT-PCR)检测大鼠尿激酶型纤溶酶原激活因子(uPA)mRNA的引物。从基因库获取靶基因及相关序列,充分收集争分析相关生物信息学数据,应用Oligo 6.22设计出一对长度为21bp的引物,其GG含量为52.4%;上下游引物3’最稳定二聚体和及发夹结构的能量分别为-1.5、-0.40 kcal/mol和-3.5、-O.90 kcal/mol,引物间最稳定二聚体为-3.1 kcal/mol。5’端和中间△G值较高,高于3’端△G;引发效率分别455和403。实验证明,该引物能够高效、特异地实现对靶序列的检测,适用于SYBR Green I法实时定量检测(uPA)mRNA。     

防治荒漠化的“三圈”生态-生产范式机理及其功能

防治荒漠化的“三圈”生态-生产范式,是干旱生态系统优化与重建的新结构。根据在鄂尔多斯和新疆等地的研究和实践,从干旱生态系统结构、过程和功能来讨论荒漠化防治的“三圈”范式。地理圈层结构（地理地带性）是“三圈”范式的自然地理背景。从宏观尺度和功能方面划分,“三圈”范式在空间尺度上有大“三圈”和小“三圈”之分,大“三圈”控制洲际范围的荒漠化扩展及沙尘暴蔓延,小“三圈”则以控制区域性风沙活动、沙尘暴和就地起沙的危害。防治荒漠化工程是复杂的多元组合和多功能的系统,我国西北地区受荒漠化影响严重,宏观的大“三圈”与多区域的小“三圈”有机结合,形成圈圈相护、层层设防的严密防护与生产系统,有效地控制大范围风沙危害、改善地方气候与小气候,并对发展经济,提高人民生活发挥重要作用。因此,“三圈”范式的概念与结构既是以自然地理地带性为基础,又是人类对自然、环境与生态系统格局的规律认识,更是科学的人类恢复、重建干旱区生态环境与可持续发展的生态设计范式。     

Boosting protein stability with the computational design of β‐sheet surfaces

β‐sheets often have one face packed against the core of the protein and the other facing solvent. Mutational studies have indicated that the solvent‐facing residues can contribute significantly to protein stability, and that the preferred amino acid at each sequence position is dependent on the precise structure of the protein backbone and the identity of the neighboring amino acids. This suggests that the most advantageous methods for designing β‐sheet surfaces will be approaches that take into account the multiple energetic factors at play including side chain rotamer preferences, van der Waals forces, electrostatics, and desolvation effects. Here, we show that the protein design software Rosetta, which models these energetic factors, can be used to dramatically increase protein stability by optimizing interactions on the surfaces of small β‐sheet proteins. Two design variants of the β‐sandwich protein from tenascin were made with 7 and 14 mutations respectively on its β‐sheet surfaces. These changes raised the thermal midpoint for unfolding from 45°C to 64°C and 74°C. Additionally, we tested an empirical approach based on increasing the number of potential salt bridges on the surfaces of the β‐sheets. This was not a robust strategy for increasing stability, as three of the four variants tested were unfolded.     

Dissecting contact potentials for proteins: relative contributions of individual amino acids

Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of this hypothesis will rely on co-crystallographic studies. Alternatively, the selection of mimotopes from a large (<4 x 10(8)) constrained library may have allowed selection of variants with even more favorable epitope topologies than present in the original antigenic structure, illustrating the power of in vivo selection of mimotopes from phage-displayed molecular libraries.     

Improving coiled-coil stability by optimizing ionic interactions

Alpha-helical coiled coils are a common protein oligomerization motif stabilized mainly by hydrophobic interactions occurring along the coiled-coil interface. We have recently designed and solved the structure of a two-heptad repeat coiled-coil peptide that is stabilized further by a complex network of inter- and intrahelical salt-bridges in addition to the hydrophobic interactions. Here, we extend and improve the de novo design of this two heptad-repeat peptide by four newly designed peptides characterized by different types of ionic interactions. The contribution of these different types of ionic interactions to coiled-coil stability are analyzed by CD spectroscopy and analytical ultracentrifugation. We show that all peptides are highly alpha-helical and two of them are 100% dimeric under physiological conditions. Furthermore, we have solved the X-ray structure of the most stable of these peptides and the rational design principles are verified by comparing this structure to the structure of the parent peptide. We show that by combining the most favorable inter- and intrahelical salt-bridge arrangements it is possible to design coiled-coil oligomerization domains with improved stability properties.     

符号检验法及其在动物生态学研究中的应用

符号检验法是非参数统计法的一种重要方法,在动物生态学的实验数据统计分析领域有一定的实用价值,本文简要介绍这一方法的使用。