Topology of wolfgram proteins and 2?, 3?-cyclic nucleotide 3?-phosphodiesterase in CNS myelin: Studies with proteases

The topological disposition of Wolfgram proteins (WP) and their relationship with 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in human, rat, sheep, bovine, guinea pig and chicken CNS myelin was investigated. Controlled digestion of myelin with trypsin gave a 35KDa protein band (WP-t) when electrophoresed on dodecyl sulfate-polyacrylamide gel in all species. Western blot analysis showed that the WP-t was derived from WP. WP-t was also formed when rat myelin was treated with other proteases such as kallikrein, thermolysin and leucine aminopeptidase. Staining for CNPase activity on nitrocellulose blots showed that WP-t is enzymatically active. Much of the CNPase activity remained with the membrane fraction even after treatment with high concentrations of trypsin when WP were completely hydrolysed and no protein bands with M.W > 14KDa were detected on the gels. Therefore protein fragments of WP with M.W < 14KDa may contain CNPase activity. From these results, it is suggested that the topological disposition of all the various WP is such that a 35KDa fragment is embedded in the lipid bilayer and the remaining fragment exposed at the intraperiod line in the myelin structure which may play a role in the initiation of myelinogenesis.

Transmembrane domains participate in functions of integral membrane proteins

Integral membrane proteins have one or more transmembrane a-helical domains and carry out a variety of functions such as enzyme catalysis, transport across membranes, transducing signals as receptors of hormones and growth factors, and energy transfer in ATP synthesis. These transmembrane domains are not mere structural units anchoring the protein to the lipid bilayer but seem to-contribute in the overall activity. Recent findings in support of this are described using some typical examples-LDL receptor, growth factor receptor tyrosine kinase, HMG-CoA reductase, F-0-ATPase and adrenergic receptors. The trends in research indicate that these transmembrane domains participate in a variety of ways such as a linker, a transducer or an exchanger in the overall functions of these proteins in transfer of materials, energy and signals.

Fusion of Complementary Online and Offline Strategies for Recognition of Handwritten Kannada Characters

This work describes an online handwritten character recognition system working in combination with an offline recognition system. The online input data is also converted into an offline image, and parallely recognized by both online and offline strategies. Features are proposed for offline recognition and a disambiguation step is employed in the offline system for the samples for which the confidence level of the classifier is low. The outputs are then combined probabilistically resulting in a classifier out-performing both individual systems. Experiments are performed for Kannada, a South Indian Language, over a database of 295 classes. The accuracy of the online recognizer improves by 11% when the combination with offline system is used.

Simultaneous purification of biotin-binding proteins-I and -II from chicken egg yolk and their characterization

Chicken egg yolk biotin-binding protein-I (BBP-I) has been purified to homogeneity along with the tetrameric BBP-II by a common protocol. The purification includes delipidation of egg yolk by butanol extraction, DEAE-Sephacel chromatography, treatment with guanidinium chloride and biotin-aminohexyl-Sepharose affinity chromatography. The identity of purified BBP-I was ascertained by its physicochemical properties as well as by its immunological cross-reactivity and precursor-product relationship with BBP-II.

Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea)

Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids. Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C-4 reversed phase HPLC chromatographic methods. In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography. The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E. coli acyl-ACP synthetase and [1-C-14]palmitic acid. When compared with E. coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions. In the absence of reducing agents formation of dimers was quite prominent. The ACPs from both the seed sources were acid- and heat-stable. The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively. However, there is significant variation in their amino acid composition. A comparision of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E. coli ACP.

Estimating the Threshold Surface Density of Gp120-CCR5 Complexes Necessary for HIV-1 Envelope-Mediated Cell-Cell Fusion

Reduced expression of CCR5 on target CD4(+) cells lowers their susceptibility to infection by R5-tropic HIV-1, potentially preventing transmission of infection and delaying disease progression. Binding of the HIV-1 envelope (Env) protein gp120 with CCR5 is essential for the entry of R5 viruses into target cells. The threshold surface density of gp120-CCR5 complexes that enables HIV-1 entry remains poorly estimated. We constructed a mathematical model that mimics Env-mediated cell-cell fusion assays, where target CD4(+)CCR5(+) cells are exposed to effector cells expressing Env in the presence of a coreceptor antagonist and the fraction of target cells fused with effector cells is measured. Our model employs a reaction network-based approach to describe protein interactions that precede viral entry coupled with the ternary complex model to quantify the allosteric interactions of the coreceptor antagonist and predicts the fraction of target cells fused. By fitting model predictions to published data of cell-cell fusion in the presence of the CCR5 antagonist vicriviroc, we estimated the threshold surface density of gp120-CCR5 complexes for cell-cell fusion as similar to 20 mu m(-2). Model predictions with this threshold captured data from independent cell-cell fusion assays in the presence of vicriviroc and rapamycin, a drug that modulates CCR5 expression, as well as assays in the presence of maraviroc, another CCR5 antagonist, using sixteen different Env clones derived from transmitted or early founder viruses. Our estimate of the threshold surface density of gp120-CCR5 complexes necessary for HIV-1 entry thus appears robust and may have implications for optimizing treatment with coreceptor antagonists, understanding the non-pathogenic infection of non-human primates, and designing vaccines that suppress the availability of target CD4(+)CCR5(+) cells.

Prediction of separation factor in foam separation of proteins

A phenomenological model has been developed for predicting separation factors obtained in concentrating protein solutions using batch-foam columns. The model considers the adsorption of surface active proteins onto the air-water interface of bubbles, and drainage of liquid from the foam, which are the two predominant processes responsible for separation in foam columns. The model has been verified with data collected on casein and bovine serum albumin (BSA) solutions, for which adsorption isotherms are available in the literature. It has been found that an increase in liquid pool height above the gas distributor and the time allowed for drainage result in a better separation. Further, taller foam columns yield poorer separation at constant time of drainage. The model successfully predicts the observed results. (C) 1997 Elsevier Science Ltd.

Synthesis and vesicle formation from dimeric pseudoglyceryl lipids with (CH2)(m) spacers: pronounced m-value dependence of thermal properties, vesicle fusion, and cholesterol complexation

Eight new dimeric lipids, in which the two Me2N+ ion headgroups are separated by a variable number of polymethylene units [-(CH2)(m)-], have been synthesized. The electron micrograph (TEM) and dynamic light scattering (DLS) of their aqueous dispersions confirmed the formation of vesicular-type aggregates. The vesicle sizes and morphologies were found to depend strongly on the m value, the method, and thermal history of the vesicle preparation. Information on the thermotropic properties of the resulting vesicles was obtained from microcalorimetry and temperature-dependent fluorescence anisotropy measurements. Interestingly, the T-m values for these vesicles revealed a nonlinear dependence on spacer chain length (m value). These vesicles were able to entrap riboflavin. The rates of permeation of the OH- ion under an imposed transmembrane pH gradient were also found to depend significantly on the m value. X-Ray diffraction of the cast films of the lipid dispersions elucidated the nature and the thickness of these membrane organizations, and it was revealed that these lipids organize in three different ways depending on the m value. The EPR spin-probe method with the doxylstearic acids 5NS, 12NS, and 16NS, spin-labeled at various positions of stearic acid, was used to establish, the chain-flexibility gradient and homogeneity of these bilayer assemblies. The apparent fusogenic propensities of these bipolar tetraether lipids were investigated in the presence of Na2SO4 with fluorescence-resonance energy-transfer fusion assay. Small unilamellar vesicles formed from 1 and three representative biscationic lipids were also studied with fluorescence anisotropy and H-1 NMR spectroscopic techniques in the absence and the presence of varying amounts of cholesterol.

A C-H center dot center dot center dot O hydrogen bond stabilized polypeptide chain reversal motif at the C terminus of helices in proteins

The serendipitous observation of a C-H...O hydrogen bond mediated polypeptide chain reversal in synthetic peptide helices has led to a search for the occurrence of a similar motif in protein structures. From a dataset of 634 proteins, 1304 helices terminating in a Schellman motif have been examined. The C-H...O interaction between the T - 4 (CH)-H-alpha and T + 1 C=O group (C...O 3.5 Angstrom) becomes possible only when the T + 1 residue adopts an extended beta conformation (T is defined as the helix terminating residue adopting an alpha(L) conformation). In all, 111 examples of this chain reversal motif have been identified and the compositional and conformational. preferences at positions T - 4, T, and T + 1 determined. A marked preference for residues like Set, Glu and Gln is observed at T - 4 position with the motif being further stabilized by the formation of a side-chain-backbone O...H-N hydrogen bond involving the side-chain of residue T - 4 and the N-H group of residue T + 3. In as many as 57 examples, the segment following the helix was extended with three to four successive residues in beta conformation. In a majority of these cases, the succeeding beta strand lies approximately antiparallel with the helix, suggesting that the backbone C-H...O interactions may provide a means of registering helices and strands in an antiparallel orientation. Two examples were identified in which extended registry was detected with two sets of C-H...O hydrogen bonds between (T - 4) (CH)-H-alpha...C=O (T + 1) and (T - 8) (CH)-H-alpha...C=O (T + 3). 0 2002 Published by Elsevier Science Ltd.

DEPTH: a web server to compute depth and predict small-molecule binding cavities in proteins

Depth measures the extent of atom/residue burial within a protein. It correlates with properties such as protein stability, hydrogen exchange rate, protein-protein interaction hot spots, post-translational modification sites and sequence variability. Our server, DEPTH, accurately computes depth and solvent-accessible surface area (SASA) values. We show that depth can be used to predict small molecule ligand binding cavities in proteins. Often, some of the residues lining a ligand binding cavity are both deep and solvent exposed. Using the depth-SASA pair values for a residue, its likelihood to form part of a small molecule binding cavity is estimated. The parameters of the method were calibrated over a training set of 900 high-resolution X-ray crystal structures of single-domain proteins bound to small molecules (molecular weight < 1.5 KDa). The prediction accuracy of DEPTH is comparable to that of other geometry-based prediction methods including LIGSITE, SURFNET and Pocket-Finder (all with Matthew's correlation coefficient of similar to 0.4) over a testing set of 225 single and multi-chain protein structures. Users have the option of tuning several parameters to detect cavities of different sizes, for example, geometrically flat binding sites. The input to the server is a protein 3D structure in PDB format. The users have the option of tuning the values of four parameters associated with the computation of residue depth and the prediction of binding cavities. The computed depths, SASA and binding cavity predictions are displayed in 2D plots and mapped onto 3D representations of the protein structure using Jmol. Links are provided to download the outputs. Our server is useful for all structural analysis based on residue depth and SASA, such as guiding site-directed mutagenesis experiments and small molecule docking exercises, in the context of protein functional annotation and drug discovery.

Phenobarbitone-mediated translocation of the cytosolic proteins interacting with the 5 '-proximal region of rat liver CYP2B1/B2 gene into the nucleus

The positive element (PE) (-69 to -98 bp) within the 5'-proximal region of the CYP2B1B2 gene (+1 to -179 bp) of rat liver is essential for phenobarbitone (PB) response and gives a single major complex with the rat liver cytosol in gel shift analysis. This complex corresponds to complex I (top) of the three complexes given by the nuclear extracts. PB treatment of rats leads to a decrease in complex I formation with the cytosol and PE and an increase in the same with the nuclear extract in gel shift analysis. Both the changes are counteracted by simultaneous okadaic acid administration. The nuclear protein giving rise to complex I has been isolated and has an M-r of 26 kDa. The cytosolic counterpart consists of two species, 26 and 28 kDa, as revealed by Southwestern blot analysis using labeled PE. It is concluded that PB treatment leads to the translocation accompanied by processing of the cytosolic protein species into the nucleus that requires protein dephosphorylation. It is suggested that PB may exert a global regulation on the transcription of many genes by modulating the phosphorylation status of different protein factors involved in transcriptional regulation. (C) 2002 Elsevier Science (USA).

The design and synthesis of redox core-alpha amino acid composites based on thiol-disulfide exchange mechanism and a comparative study of their zinc abstraction potential from [CCXX] boxes in proteins

The design and synthesis of agents that can abstract zinc from their [CCXX] (C=cysteine; X=cysteine/histidine) boxes by thioldisulfide exchange-having as control, the redox parities of the core sulfur ligands of the reagent and the enzyme, has been illustrated, and their efficiency demonstrated by monitoring the inhibition of the transcription of calf thymus DNA by E. coli RNA polymerase, which harbors two zinc atoms in their [CCXX] boxes of which one is exchangeable. Maximum inhibition possible with removal of the exchangeable zinc was seen with redox-sulfanilamide-glutamate composite. In sharp contrast, normal chelating agents (EDTA, phenanthroline) even in a thousand fold excess showed only marginal inhibition, thus supporting an exchange mechanism for the metal removal. (C) 2002 Elsevier Science Ltd. All rights reserved.