RESUMEN
A leucine-rich repeat plant protein involved in resistance to pathogens, a polygalacturonase-inhibiting protein (PGIP-1) from Phaseolus vulgaris, has been crystallized and preliminary X-ray characterization has been performed. The protein contains ten repeats of a short (24 amino-acid) leucine-rich repeat motif. Single crystals of the protein were grown from vapour-diffusion experiments using PEG 2K monomethylether as precipitant; these crystals diffract to at least 2.3 A resolution. The space group is P2(1), with two molecules of PGIP-1 in the asymmetric unit; the crystals contain approximately 38% solvent.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Fabaceae/química , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Plantas Medicinales , Poligalacturonasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Fabaceae/genética , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Two members of the pgip gene family (pgip-1 and pgip-2) of Phaseolus vulgaris L. were expressed separately in Nicotiana benthamiana and the ligand specificity of their products was analysed by surface plasmon resonance (SPR). Polygalacturonase-inhibiting protein-1 (PGIP-1) was unable to interact with PG from Fusarium moniliforme and interacted with PG from Aspergillus niger; PGIP-2 interacted with both PGs. Only eight amino acid variations distinguish the two proteins: five of them are confined within the beta-sheet/beta-turn structure and two of them are contiguous to this region. By site-directed mutagenesis, each of the variant amino acids of PGIP-2 was replaced with the corresponding amino acid of PGIP-1, in a loss-of-function approach. The mutated PGIP-2s were expressed individually in N.benthamiana, purified and subjected to SPR analysis. Each single mutation caused a decrease in affinity for PG from F.moniliforme; residue Q253 made a major contribution, and its replacement with a lysine led to a dramatic reduction in the binding energy of the complex. Conversely, in a gain-of-function approach, amino acid K253 of PGIP-1 was mutated into the corresponding amino acid of PGIP-2, a glutamine. With this single mutation, PGIP-1 acquired the ability to interact with F.moniliforme PG.
Asunto(s)
Proteínas de Plantas/química , Poligalacturonasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Fabaceae/genética , Fusarium/enzimología , Biblioteca de Genes , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/genética , Plantas Medicinales , Plantas Tóxicas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Secuencias Repetitivas de Aminoácido , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Nicotiana/genéticaRESUMEN
A ginseng polypeptide (GPP) found in ginseng roots and its modified peptides were tested by capillary zone electrophoresis (CZE) under acidic as well as basic conditions. Modified peptides were synthesized for three purposes: (i) to analyze their functions in the first three acidic amino acid residues, (ii) to analyze their functions in three sequenced glycines, and (iii) to analyze the length of side chains in acidic amino acids. The roles of glycines, acidic amino acids and amino acid side chains in the binding of Mg2+ were studied at pH values less than 7.0. The migration times of GPP varied with the pH of various electrophoresis buffers, and electrophoresis patterns were significantly changed between pH 7.0-7.5. Based on the electrophoretic analysis, it was concluded that the binding mechanisms for Mg2+ or conformations of GPP changed between low pH and high pH.
Asunto(s)
Electroforesis Capilar/métodos , Panax/química , Proteínas de Plantas/análisis , Plantas Medicinales , Tampones (Química) , Estructura Molecular , Péptidos/análisis , Fosfatos , Acetato de Sodio , TrometaminaRESUMEN
A tetradecapeptide from ginseng (Panax ginseng) root showing anti-lipolytic activity in an isolated rat fat cell assay was chemically synthesized for analysis of metal binding activities in vitro. Binding activities against several metal ions were analysed by measuring mobility shifts during capillary zone electrophoresis experiments. The ginseng polypeptide (GPP) showed the greatest increase in effective molecular electrophoretic mobility in the presence of Mg2+. Mobility was also affected in the presence of La3+, Mn2+, Ca2+ and Zn2+ ions. Analysis with the dye Stains-all revealed GPP to possess a cation binding site similar to those in Ca(2+)-binding proteins. GPP thus appears to be a metal binding peptide. The results of this analysis suggested that GPP may perform its anti-lipolytic activities through an ability to modulate the level of free cellular Mg2+ and Mn2+ ions.