One step purification of peanut phospholipase D by precipitation with alginate

A simple titrimetric assay with soybean lecithin has been used for screening phospholipase D activity from some plant sources, viz. peanut, wheat germ, cabbage and carrot. The enzyme from peanut has been purified by binding to alginate which is a water soluble polymer. The purification consisted of co-precipitation of enzyme with alginate upon addition of 0.06 M Ca⁺⁺. The enzyme was eluted from the polymer using 0.2 M sodium chloride. The activity recovery was 61% with 34 fold purification.     

Effects of diquat on pigment-protein complexes of thylakoid membranes in soybean and maize plants

Soybean (Glycine max Merrill) and maize (Zea mays L.) plants were exposed for 5 to 48 h to the herbicide diquat under "white light" (WL) or far-red radiation (FR) (photon fluence rate of 30 μmol m^-2 s^-1). The WL enhanced diquat effect on chlorophyll content in soybean plants, while FR had the same effects on maize plants. After 5 h, diquat increased the content of polypeptides bound to light-harvesting proteins in both plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Association of Tau-Like Proteins with Vimentin Filaments in Cultured Cells

There is increasing evidence that the different polymers that constitute the cytoskeleton are interconnected to form a three-dimensional network. The macromolecular interaction patterns that stabilize this network and its intrinsic dynamics are the basis for numerous cellular processes. Within this context,in vitrostudies have pointed to the existence of specific associations between microtubules, microfilaments, and intermediate filaments. It has also been postulated that microtubule-associated proteins (MAPs) are directly involved in mediating these interactions. The interactions of tau with vimentin filaments, and its relationships with other filaments of the cytoskeletal network, were analyzed in SW-13 adenocarcinoma cells, through an integrated approach that included biochemical and immunological studies. This cell line has the advantage of presenting a wild-type clone (vim+) and a mutant clone (vim−) which is deficient in vimentin expression. We analyzed the cellular roles of tau, focusing on its interactions with vimentin filaments, within the context of its functional aspects in the organization of the cytoskeletal network. Cosedimentation experiments of microtubular protein with vimentin in cell extracts enriched in intermediate filaments, combined with studies on the direct interaction of tau with nitrocellulose-bound vimentin and analysis of tau binding to vimentin immobilized in single-strand DNA affinity columns, indicate that tau interacts with the vimentin network. These studies were confirmed by a quantitative analysis of the immunofluorescence patterns of cytoskeleton-associated tubulin, tau, and vimentin using flow cytometry. In this regard, a decrease in the levels of tau associated to the cytoskeletal network in the vim− cell mutant compared with the wild-type clones was observed. However, immunofluorescence data on SW-13 cells suggest that the absence of a structured network of vimentin in the mutant vim− cells does not affect the cytoplasmic organization formed by microtubules and actin filaments, when compared with the wild-type vim+ cells. These studies suggest that tau associates with vimentin filaments and that these interactions may play a structural role in cells containing these filaments.     

Oxidative folding of synthetic polypeptides S‐protected as tert‐butylthio derivatives

A new method for oxidative folding of synthetic polypeptides assembled by stepwise solid phase synthesis is introduced. Folding is obtained in excellent yields by reacting S‐tert‐butylthiolated polypeptides with a 100‐fold molar excess of cysteine at 37 °C in a slightly alkaline buffer containing chaotropic salts, and in the presence of air‐oxygen. This novel protocol has been applied to the folding of S‐tert‐butylthiolated human thymus and activation‐regulated chemokine (hu‐TARC) derivatives as well as to larger segments of Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Folded P. falciparum polypeptides have been used as substrates of endoproteinase Glu‐C (Glu‐C) and endoproteinase Asp‐N (Asp‐N) in an attempt to identify their disulfide connectivities. Particular practical advantages of the present method are (i) easy purification and storage of the S‐protected peptide derivatives, (ii) elimination of the risk of cysteine alkylation during the acidolytic cleavage deprotection and resin cleavage steps, (iii) possibility to precisely evaluate the extent of folding and disulfide bond formation by mass spectrometry, and (iv) facile recovery of the final folded product. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

类弹性蛋白多肽及其在生物医学材料的应用

类弹性蛋白多肽是一种人造基因工程蛋白质聚合物,其结构主要由五肽重复串连序列单元 (GVGXP) 的这一肽段单元重复组成。由于具有可逆相变特征,并可进行高通量生产,加之良好的生物相容性及生物可降解性,使其在新型生物医学材料方面展示了广阔的应用前景。概括了类弹性蛋白多肽的相变机理、合成方法及在生物医学材料上的应用,重点阐述了类弹性蛋白多肽在组织工程、靶向肿瘤、构造药物载体微粒的应用。     

Microsomal polypeptides in sunflower (Helianthus annum). Comparison between normal type before and after cold-induction, and a high oleic acid mutant

Differences in high-resolution two-dimensional gel electrophoresis patterns of micro-somal proteins from developing normal sunflower ( Helianthus annuus L.) seeds before and after cold-induction, and also from normal and a high oleic sunflower mutant have been studied in order to detect the polypeptides associated with the microsomal Δ^l2-desaturase activity and its regulation by temperature. Proteins were obtained from developing seeds of two isogenic sunflower lines HA-89 (normal) and HA-OL9 (high oleic) which greatly differed in linoleic acid content and "in vitro" oleate desaturase activity. In the high oleic mutant, four polypeptides of about 32 kDa and two of 33 kDa were found to change in position, to the same extent, toward a lower isoelectric point in the high oleic mutant. Also, two polypeptides, of 32 and 49 kDa each, appeared in the mutant. Quantitative differences between cold-induced seeds (10°C, 24 h) and their non-induced controls were found. One polypeptide of 43 kDa decreased in the cold-treated seeds and two others, of 30 and 32 kDa each, increased markedly after cold induction. Some of these polypeptides could be related to oleate desatnrase activity or its regulation by temperature.     

Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S]^1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S]^1+,2+ clusters termed F_A and F_B. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F_B. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S]^1+,2+ clusters, despite the absence of one of the biological ligands. ¹⁹F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F_B cluster. The finding that site-modified [4Fe-4S]^1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.     

Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa

The fluorescent protein Dronpa undergoes reversible photoswitching reactions between the bright “on” and dark “off” states via photoisomerization and proton transfer reactions. We report the room temperature crystal structure of the fast switching Met159Thr mutant of Dronpa at 2.0‐Å resolution in the bright on state. Structural differences with the wild type include shifted backbone positions of strand β8 containing Thr159 as well as an altered A‐C dimer interface involving strands β7, β8, β10, and β11. The Met159Thr mutation increases the cavity volume for the p‐hydroxybenzylidene‐imidazolinone chromophore as a result of both the side chain difference and the backbone positional differences. Proteins 2015; 83:397–402. © 2014 Wiley Periodicals, Inc.