Polymorphisms in the Human Tropoelastin Gene Modify In Vitro Self-Assembly and Mechanical Properties of Elastin-Like Polypeptides

Elastin is a major structural component of elastic fibres that provide properties of stretch and recoil to tissues such as arteries, lung and skin. Remarkably, after initial deposition of elastin there is normally no subsequent turnover of this protein over the course of a lifetime. Consequently, elastic fibres must be extremely durable, able to withstand, for example in the human thoracic aorta, billions of cycles of stretch and recoil without mechanical failure. Major defects in the elastin gene (ELN) are associated with a number of disorders including Supravalvular aortic stenosis (SVAS), Williams-Beuren syndrome (WBS) and autosomal dominant cutis laxa (ADCL). Given the low turnover of elastin and the requirement for the long term durability of elastic fibres, we examined the possibility for more subtle polymorphisms in the human elastin gene to impact the assembly and long-term durability of the elastic matrix. Surveys of genetic variation resources identified 118 mutations in human ELN, 17 being non-synonymous. Introduction of two of these variants, G422S and K463R, in elastin-like polypeptides as well as full-length tropoelastin, resulted in changes in both their assembly and mechanical properties. Most notably G422S, which occurs in up to 40% of European populations, was found to enhance some elastomeric properties. These studies reveal that even apparently minor polymorphisms in human ELN can impact the assembly and mechanical properties of the elastic matrix, effects that over the course of a lifetime could result in altered susceptibility to cardiovascular disease.     

Molecular Weight Determination of Component Polypeptides of Glutenin after Fractionation by Gel Filtration

Reduced and cyanoethylated glutenin was fractionated into three fractions (F I, F II and F III) by gel filtration on Sephadex G–100 in 0.1 m acetic acid. The molecular weight determination was made with these three fractions by sedimentation equilibrium in 6.5 m guanidine hydrochloride containing 0.01 m acetic acid. The molecular weight obtained was 44,000 for F II, and 32,000 for F III. F I showed a distribution of molecular weight due to the aggregation. The average molecular weight of F I was 52,000, being 27,000 at the meniscus and 98,000 at the bottom. The estimation of molecular weight by SDS–PAGE* gave overestimated values for glutenin polypeptides, as was already reported for gliadin.     

Molecular Interlayers in Hybrid Perovskite Solar Cells

Organic–inorganic hybrid perovskite solar cells (PSC) are promising third‐generation solar cells. They exhibit good power conversion efficiencies and in principle they can be fabricated with lower energy consumption than many more established technologies. To improve the efficiency and long‐term stability of PSC, organic molecules are frequently used as “interlayers.” Interlayers are thin layers or monolayers of organic molecules that modify a specific interface in the solar cell. Here, the latest progress in the use of interlayers to optimize the performance of PSC is reviewed. Where appropriate interesting examples from the field of organic photovoltaics (OPV) are also presented as there are many similarities in the types of interlayers that are used in PSC and OPV. The review is organized into three parts. The first part focuses on why organic molecule interlayers improve the performance of the solar cells. The second section discusses commonly used molecular interlayers. In the last part, different approaches to make thin and uniform interlayers are discussed.     

Molecular Dynamic Simulation of the Self-Assembly of DAP12-NKG2C Activating Immunoreceptor Complex

The DAP12-NKG2C activating immunoreceptor complex is one of the multisubunit transmembrane protein complexes in which ligand-binding receptor chains assemble with dimeric signal-transducing modules through non-covalent associations in their transmembrane (TM) domains. In this work, both coarse grained and atomistic molecular dynamic simulation methods were applied to investigate the self-assembly dynamics of the transmembrane domains of the DAP12-NKG2C activating immunoreceptor complex. Through simulating the dynamics of DAP12-NKG2C TM heterotrimer and point mutations, we demonstrated that a five-polar-residue motif including: 2 Asps and 2 Thrs in DAP12 dimer, as well as 1 Lys in NKG2C TM plays an important role in the assembly structure of the DAP12-NKG2C TM heterotrimer. Furthermore, we provided clear evidences to exclude the possibility that another NKG2C could stably associate with the DAP12-NKG2C heterotrimer. Based on the simulation results, we proposed a revised model for the self-assembly of DAP12-NKG2C activating immunoreceptor complex, along with a plausible explanation for the association of only one NKG2C with a DAP12 dimer.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

Molecular Evidence for the Hybrid Origin of Ilex dabieshanensis (Aquifoliaceae)

Ilex, the largest genus of dioecious woody plants, is a good study system to assess the role of hybridization in speciation and evolution. Ilex dabieshanensis, a tree endemic to Dabieshan Mountains region, was initially described as a new species. Based on morphological intermediacy and sympatric distribution with its putative parental species, I. cornuta and I. latifolia, we proposed it as a natural hybrid between them. In this study, we sequenced one chloroplast intergenic spacer (trnH-psbA) and two nuclear genes (gapC and nepGS) in I. dabieshanensis and its putative parental species to test the hybrid origin hypothesis. Our results showed that there were one to two differentially fixed sequence differences between I. cornuta and I. latifolia at the two nuclear genes. Twelve of the 14 individuals of I. dabieshanensis exhibited additivity in chromatograms on these differentially fixed sites at both nuclear genes, and the remaining two exhibited additivity in chromatograms on the fixed site at only the nepGS gene. Except one haplotype of I. cornuta at the nepGS gene, all of the haplotypes of I. cornuta at the two nuclear genes were well separated from those of I. latifolia, and most haplotypes of I. dabieshanensis were shared with those of I. cornuta and I. latifolia. Phylogenetic analysis of these haplotypes was largely consistent with haplotype network analysis. I. cornuta and I. latifolia differed by two nucleotide substitutions in the chloroplast intergenic spacer, and 12 individuals of I. dabieshanensis had the same sequences as I. latifolia, while the remaining two were identical with I. cornuta. The molecular data provide convincing evidence for the hybrid origin of I. dabieshanensis and asymmetrical direction of hybridization. One haplotype of I. cornuta at the nepGS gene was nested with those of I. latifolia, indicating introgression to I. cornuta.     

A Novel Molecular Design for a Hybrid Phage-DNA Construct Against DKK1

Nucleic acid immunization has recently exhibited a great promise for immunotherapy of various diseases. However, it is now clear that powerful strategies are imminently needed to improve their efficiency. In this regard, whole bacteriophage particles have been described as efficient DNA vaccine delivery vehicles, capable of circumventing the limitations of naked DNA immunization. Moreover, phage particles could be engineered to display specific peptides on their surfaces. Given these inherent characteristics of phages, we have designed a novel hybrid phage-DNA immunization vector using both M13 and pAAV plasmid elements. Following the construction and in vitro confirmation of the designed vectors, they were used for comparative mice immunization, carrying the same DNA sequence. The results indicated the efficacy of the designed hybrid phage particles, to elicit higher humoral immunity, in comparison to conventional DNA-immunization vectors (pCI). In light of these findings, it could be concluded that using adeno-associated virus (AAV) expression cassette along with displaying TAT peptide on the surface of the phage particle could be deemed as an appealing strategy to enhance the DNA-immunization and vaccination efficacy.     

A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (V_H) and light (V_L) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (V_L-V_H) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli.     

Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.l-Arabinose isomerase (l-AI) is an enzyme that mediates in vivo isomerization between l-arabinose and l-ribulose as well as in vitro isomerization of d-galactose and d-tagatose (20). l-Ribulose (l-erythro-pentulose) is a rare and expensive ketopentose sugar (1) that can be used as a precursor for the production of other rare sugars of high market value, such as l-ribose. Despite being a common metabolic intermediate in different organisms, l-ribulose is scarce in nature. The market for rare and unnatural sugars has been growing, especially in the sweetener and pharmaceutical industries. For example, several modified nucleosides derived from l-sugars have been shown to act as potent antiviral agents and are also useful in antigen therapy. Derivatives of rare sugars have also been used as agents against hepatitis B virus and human immunodeficiency virus (2, 22).For these reasons, interest in the enzymology of rare sugars has also been increasing. Various forms of l-AI from a variety of organisms have been characterized, and some have shown potential for industrial use. Several highly thermotolerant enzyme forms from Thermotoga maritima (12), Thermotoga neapolitana (10), Bacillus stearothermophilus (18), Thermoanaerobacter mathranii (9), and Lactobacillus plantarum (5) have been characterized previously. All of these reported l-AIs tend to have broad specificity, although a few l-AIs with high degrees of substrate specificity for l-arabinose have also been documented.The enzyme properties of l-AIs have been examined by engineering several forms by error-prone PCR and site-directed mutagenesis. Galactose conversion was reportedly enhanced 20% following site-directed introduction of a double mutation (C450S-N475K) into l-AI (16). Error-prone PCR manipulation of l-AI from Geobacillus stearothermophilus resulted in a shift in temperature specificity from 60 to 65°C and increased isomerization activity (11). All of these previously reported mutational studies have been aimed at improving enzymatic properties for industrial application. However, even though the three-dimensional (3D) structure of Escherichia coli l-AI has been determined previously (15), few new structural studies have been performed to decipher the reaction mechanism of this enzyme. Rhimi et al. (19) have reported an important role for D308, F329, E351, and H446 in catalysis, as indicated by findings from site-directed mutagenesis. Nonetheless, detailed analysis of the important molecular determinants controlling the catalytic activities of the l-AIs is still lacking.Previously, we have reported the cloning and characterization of a novel l-AI from Bacillus licheniformis (17). This enzyme can be distinguished from other l-AIs by its wide pH range, high degree of substrate specificity for l-arabinose, and extremely high turnover rate. In the present paper, we report the identification of an important amino acid residue responsible for the catalytic efficiency of l-AIs, as determined by a systematic screening process composed of sequence alignment and molecular dynamics (MD) simulation, followed by site-directed mutagenesis. Using the crystal structure of E. coli l-AI as a template, we have built a 3D model of B. licheniformis l-AI. Analysis of the 3D model of B. licheniformis l-AI docked with l-arabinose, followed by a systematic screening process, showed that Y333 interacted with the substrate, suggesting that this residue in B. licheniformis l-AI may be essential for catalysis. We further characterized the role of Y333 in B. licheniformis l-AI binding of and catalytic efficiency for l-arabinose.     

Simple Moment-Closure Model for the Self-Assembly of Breakable Amyloid Filaments

In this work, we derive a simple mathematical model from mass-action equations for amyloid fiber formation that takes into account the primary nucleation, elongation, and length-dependent fragmentation. The derivation is based on the principle of minimum free energy under certain constraints and is mathematically related to the partial equilibrium approximation. Direct numerical comparisons confirm the usefulness of our simple model. We further explore its basic kinetic and equilibrium properties, and show that the current model is a straightforward generalization of that with constant fragmentation rates.     

A Strategy for the Molecular Diagnosis in Hemophilia A in Chinese Population

Hemophilia A is an x-linked recessive inherited bleeding disorder. So far, more than 1,885 disease-causing mutations of factor VIII gene have been identified. Clinic confers a great challenge for the molecular diagnosis. We aim to make a better strategy for the molecular diagnosis in Hemophilia A. First, factor VIII intron 22 inversion and intron 1 inversion mutations were detected using Inversion-PCR and double-tube multiple PCRs. And then, non-inversion mutations were analyzed by denaturing high performance liquid chromatography and/or direct sequencing. Novel mutations were further analyzed the conservation and 3D structures by a B domain deleted crystallographic model and bioinformatics. Finally, we can indirectly confirm the diagnosis by linkage analysis for the patients with the confusing diagnosis by the techniques mentioned above. Eleven patients with the factor VIII Inv 22 were found, and the remaining 16 patients were found with 11 different mutations, of which 3 was novel mutations affecting A1, B domains and splicing site. Moreover, the prenatal diagnosis was performed on 14 fetuses. Ten fetuses were successfully confirmed to be normal, 1 fetus to be a heterozygote with factor VIII c.3275–3276 ins A and 3 fetuses to be hemizygotes with factor VIII Inv 22 mutation.     

Anti-Fading Agents for Confocal Immunofluorescence: Colocalization of Nuclear Polypeptides

We evaluated the performance of four anti-fading agents during acquisition of multiple optical sections near the widest diameter of Drosophila accessory gland nuclei using indirect immunofluorescence and confocal laser scanning microscopy. Two commercially available agents, Vectashield® and SlowFade® showed anti-fading properties that alleviated fluorochrome fading associated with the acquisition of multiple fluorescent optical Z-series from a single specimen by a confocal laser scanning system. Using these reagents, we were able to colocalize polypeptides through immunostained whole Drosophila nuclei.     

HOLLOW: Generating Accurate Representations of Channel and Interior Surfaces in Molecular Structures

Background  

An accurate rendering of interior surfaces can facilitate the analysis of mechanisms at atomic-level detail, such as the transport of substrates in the ammonia channel. In molecular viewers, one must remove the exterior surface that obscures the channel surface by clipping the viewing plane or manually selecting the channel residues in order to display a partial surface. Neither method is entirely satisfactory, as unwanted additional pieces of surfaces are always generated.     

我国杂交水稻基因工程育种策略探讨

杂交水稻在我国水稻及粮食生产中占有突出地位 ,基因工程可在杂交水稻育种中发挥重要作用。针对杂交水稻育种存在的主要问题 ,指出应将优质、抗虫和抗病作为当前基因工程育种的研究重点。同时提出基因工程育种与常规育种紧密结合 ,优质基因工程着重改良保持系及聚合转基因等策略 ,以培育出超高产优质抗病虫转基因聚合杂交稻新组合。     

Molecular Modeling of Proteins: A Strategy for Energy Minimization by Molecular Mechanics in the AMBER Force Field

Abstract

Energy minimization is an important step in molecular modeling of proteins. In this study, we sought to develop a minimization strategy which would give the best final structures with the shortest computer time in the AMBER force field. In the all-atom model, we performed energy minimization of the melittin (mostly α-helical) and cardiotoxin (mostly β-sheet and β-turns) crystal structures by both constrained and unconstrained pathways. In the constrained path, which has been recommended in the energy minimization of proteins, hydrogens were relaxed first, followed by the side chains of amino acid residues, and finally the whole molecule. Despite the logic of this approach, however, the structures minimized by the unconstrained path fit the experimental structures better than those minimized by constrained paths. Moreover, the unconstrained path saved considerable computer time. We also compared the effects of the steepest descents and conjugate gradients algorithms in energy minimization. Previously, steepest descents has been used in the initial stages of minimization and conjugate gradients in the final stages of minimization. We therefore studied the effect on the final structure of performing an initial minimization by steepest descents. The structures minimized by conjugate gradients alone resembled the structures minimized initially by the steepest descents and subsequently by the conjugate gradients algorithms. Thus an initial minimization using steepest descents is wasteful and unnecessary, especially when starting from the crystal structure. Based on these results, we propose the use of an unconstrained path and conjugate gradients for energy minimization of proteins. This procedure results in low energy structures closer to the experimental structures, and saves about 70–80% of computer time. This procedure was applied in building models of lysozyme mutants. The crystal structure of native T₄ lysozyme was mutated to three different mutants and the structures were minimized. The minimized structures closely fit the crystal structures of the respective mutants (< 0.3 Å root-mean-square, RMS, deviation in the position of all heavy atoms). These results confirm the efficiency of the proposed minimization strategy in modeling closely related homologs. To determine the reliability of the united atom approximation, we also performed all of the above minimizations with united atom models. This approximation gave structures with similar but slightly higher RMS deviations than the all-atom model, but gave further savings of60-70% in computer time. However, we feel further investigation is essential to determine the reliability of this approximation. Finally, to determine the limitation of the procedure, we built the melittin molecule interactively in an α-helical conformation and this model showed an RMS deviation greater than 2.8 Å when compared to the melittin crystal structure. This model was minimized by various strategies. None of the minimized structures converged towards the crystal structure. Thus, although the proposed method seems to give valid structures starting from closely related crystal structures, it cannot predict the native structure when the starting structure is far from the native structure. From these results, we recommend the use of the proposed strategy of minimizing by an unconstrained path using the conjugate gradients algorithm, but only for modeling of closely related structural homologs of proteins.