Clusters in alpha/beta barrel proteins: implications for protein structure, function, and folding: a graph theoretical approach

The alpha/beta barrel fold is adopted by most enzymes performing a variety of catalytic reactions, but with very low sequence similarity. In order to understand the stabilizing interactions important in maintaining the alpha/beta barrel fold, we have identified residue clusters in a dataset of 36 alpha/beta barrel proteins that have less than 10% sequence identity within themselves. A graph theoretical algorithm is used to identify backbone clusters. This approach uses the global information of the nonbonded interaction in the alpha/beta barrel fold for the clustering procedure. The nonbonded interactions are represented mathematically in the form of an adjacency matrix. On diagonalizing the adjacency matrix, clusters and cluster centers are obtained from the highest eigenvalue and its corresponding vector components. Residue clusters are identified in the strand regions forming the beta barrel and are topologically conserved in all 36 proteins studied. The residues forming the cluster in each of the alpha/beta protein are also conserved among the sequences belonging to the same family. The cluster centers are found to occur in the middle of the strands or in the C-terminal of the strands. In most cases, the residues forming the clusters are part of the active site or are located close to the active site. The folding nucleus of the alpha/beta fold is predicted based on hydrophobicity index evaluation of residues and identification of cluster centers. The predicted nucleation sites are found to occur mostly in the middle of the strands. Proteins 2001;43:103-112.     

Effect of endothelial cell polarity on beta-amyloid-induced migration of monocytes across normal and AD endothelium

During normal aging and amyloid beta-peptide (Abeta) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Abeta accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Abeta(1--40) with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.     

Male gametophyte development in bread wheat (Triticum aestivum L.): molecular,cellular, and biochemical analyses of a sporophytic contribution to pollen wall ontogeny

Bread wheat (hexaploid AABBDD genome; 16 billion basepairs) is a genetically complex, self-pollinating plant with bisexual flowers that produce short-lived pollen. Very little is known about the molecular biology of its gametophyte development despite a longstanding interest in hybrid seeds. We present here a comprehensive characterization of three apparently homeologous genes (TAA1a, TAA1b and TAA1c) and demonstrate their anther-specific biochemical function. These eight-exon genes, found at only one copy per haploid complement in this large genome, express specifically within the sporophytic tapetum cells. The presence of TAA1 mRNA and protein was evident only at specific stages of pollen development as the microspore wall thickened during the progression of free microspores into vacuolated-microspores. This temporal regulation matched the assembly of wall-impregnated sporopollenin, a phenylpropanoid-lipid polymer containing very long chain fatty alcohols (VLCFAlc), described in the literature. Our results establish that sporophytic genes contribute to the production of fatty alcohols: Transgenic expression of TAA1 afforded production of long/VLCFAlc in tobacco seeds (18 : 1; 20 : 1; 22 : 1; 24 : 0; 26 : 0) and in Escherichia coli (14 : 0; 16 : 0; 18 : 1), suggesting biochemical versatility of TAA1 with respect to cellular milieu and substrate spectrum. Pollen walls additionally contain fatty alcohols in the form of wax esters and other lipids, and some of these lipids are known to play a role in the highly specific sexual interactions at the pollen-pistil interface. This study provides a handle to study these and to manipulate pollen traits, and, furthermore, to understand the molecular biology of fatty alcohol metabolism in general.     

Influence of medium- and long-range interactions in different folding types of globular proteins

Recognition of protein fold from amino acid sequence is a challenging task. The structure and stability of proteins from different fold are mainly dictated by inter-residue interactions. In our earlier work, we have successfully used the medium- and long-range contacts for predicting the protein folding rates, discriminating globular and membrane proteins and for distinguishing protein structural classes. In this work, we analyze the role of inter-residue interactions in commonly occurring folds of globular proteins in order to understand their folding mechanisms. In the medium-range contacts, the globin fold and four-helical bundle proteins have more contacts than that of DNA-RNA fold although they all belong to all-alpha class. In long-range contacts, only the ribonuclease fold prefers 4-10 range and the other folding types prefer the range 21-30 in alpha/beta class proteins. Further, the preferred residues and residue pairs influenced by these different folds are discussed. The information about the preference of medium- and long-range contacts exhibited by the 20 amino acid residues can be effectively used to predict the folding type of each protein.     

Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family,is essential for tapetum differentiation and male fertility

Membrane-bound glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) mediates the initial step of glycerolipid biosynthesis in the extraplastidic compartments of plant cells. Here, we report the molecular characterization of a novel GPAT gene family from Arabidopsis, designated AtGPAT. The corresponding polypeptides possess transmembrane domains and GPAT activity when expressed heterologously in a yeast lipid mutant. The functional significance of one isoform, AtGPAT1, is the focus of the present study. Disruption of the AtGPAT1 gene causes a massive pollen development arrest, and subsequent introduction of the gene into the mutant plant rescues the phenotype, illustrating a pivotal role for AtGPAT1 in pollen development. Microscopic examinations revealed that the gene lesion results in a perturbed degeneration of the tapetum, which is associated with altered endoplasmic reticulum profiles and reduced secretion. In addition to the sporophytic effect, AtGPAT1 also exerts a gametophytic effect on pollen performance, as the competitive ability of a pollen grain to pollinate is dependent on the presence of an AtGPAT1 gene. Deficiency in AtGPAT1 correlates with several fatty acid composition changes in flower tissues and seeds. Unexpectedly, however, a loss of AtGPAT1 causes no significant change in seed oil content.     

High-frequency plant regeneration from cotyledon callus of <Emphasis Type="Italic">Acacia sinuata</Emphasis> (Lour.) Merr

Summary A new reliable protocol for the induction of adventitious shoots and plant regenertion from cotyledon-derived callus of Acacia sinuata has been developed. Calluses were induced from cotyledon explants on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar or 0.15% phytagel, 8.1 μM α-naphthaleneacetic acid, and 2.2 μM 6-benzylaminopurine (BA). High-frequency regeneration of adventitious buds from callus was achieved when cultured on half-strength MS medium supplemented with 10% coconut water, 13.3 μM BA, and 2.5 μM zeatin. Histological studies revealed that the regenerated shoots originated from the callus. Among the various carbohydrates tested, sucrose at 87.6 mM was optimum for shoot-bud induction. Addition of 1.7 μM gibberellic acid along with 4.4 μM favored shoot elongation. In vitro-raised shoots produced prominent roots when transferred to half-strength MS medium supplemented with 7.4 μM indole-3-butyric acid. Rooted plants, thus developed, were hardened and successfully established in soil (45%). This protocol yielded an average of 40 plantlets per cotyledon explant over a period of 3 mo.     

A thin-film electrochemical study of the "blue" copper proteins,auracyanin A and auracyanin B,from the photosynthetic bacterium <Emphasis Type="Italic">Chloroflexus</Emphasis><Emphasis Type="Italic">aurantiacus</Emphasis>: the reduction potential as a function of pH

The reversible formal potentials of auracyanin A and auracyanin B, two closely related "blue" copper proteins from the photosynthetic bacterium Chloroflexus aurantiacus, have been determined by protein film voltammetry in the range 4相似文献