Jean Garnier

GOR V server for protein secondary structure prediction

SUMMARYnWe have created the GOR V web server for protein secondary structure prediction. The GOR V algorithm combines information theory, Bayesian statistics and evolutionary information. In its fifth version, the GOR method reached (with the full jack-knife procedure) an accuracy of prediction Q3 of 73.5%. Although GOR V has been among the most successful methods, its online unavailability has been a deterrent to its popularity. Here, we remedy this situation by creating the GOR V server.

Improvements in a secondary structure prediction method based on a search for local sequence homologies and its use as a model building tool

This report describes an optimised version of a secondary structure prediction method based on local homologies, using a new data base. A 63% prediction accuracy, for three states, was obtained after elimination of the protein to be predicted and all proteins with a percentage identity greater than 22% from the data base. This corresponds to a 5% increase in accuracy on the original method (Levin et al. FEBS Lett. 205 (1986) 303-308). The flexibility of the method to the incorporation of information extraneous to the prediction was demonstrated by the prediction of the homologous proteins in the data base. Using the percentage identity with the protein to be predicted, to weight the relative importance of each protein, for all proteins with a percentage identity greater than 30%, the mean correct prediction per chain was 87%. As a result this algorithm can be used during the molecular modelling process, both to give an idea of the structural similarity between two proteins and as an aid in the determination of the best alignment. Incorporation of the result of a protein folding type assignment based on the global amino-acid composition increased the overall prediction to 66%.

Towards understanding the glycoprotein hormone receptors.

Lutropin (LH), follitropin (FSH) and thyrotropin (TSH), as well as choriogonadotropin (CG, which binds to the LH receptor) constitute the glycoprotein hormone family. Their 3 receptors have been cloned during the last few months. They belong to the large group of G-protein coupled membrane proteins, with their specific N-terminal domain likely to bind the hormone and the characteristic 7 membrane-spanning segments in their C-terminal moiety. The present review discusses the main results of amino acid sequence analysis performed on the glycoprotein hormone receptors. The putative extracellular head exhibits less than 45% homology over the 3 receptors, while approximately 70% residue conservation is found in the transmembrane moiety. Here only, limited sequence homologies (approximately 20%) can be found with other G-protein coupled receptors. The secondary structure predictions performed on the 3 receptors revealed that the polypeptide sequence predicted as ordered (either alpha-helix or beta-strand) were repeated evenly throughout the extracellular head with a period of approximately 25 amino acids. This analysis helped to define the intervening loops between this ordered stretches as potential candidates for bearing at least part of the binding site of the hormones. Some of the perspectives opened by the cloning of the receptors are described, like the production of the extracellular head of the porcine LH receptor in baculovirus-infected insect cells, and the exploration of the LH receptors mechanism of functioning as a dimer.

Structure of the casein micelle. The accessibility of the subunits to various reagents

Carboxypeptidase A (CPA, mol. wt = 34600) and 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride, mol. wt = 270) were shown to react with the 3 casein subunits, α s1 -, β- and κ-caseins, which constitute the micelle. Virtually all of the C-terminal amino acids were released by CPA from these subunits without disrupting the micellar structure. It was verified that the rate of dissociation of the micelle into low molecular weight complexes is considerably slower than the rates at which these reagents act on micellar components. It is concluded that dansyl chloride, CPA, and probably also rennin, whose molecular weight is similar to that of CPA, are able to penetrate to the interior of the micelles. This was confirmed in a quite independent way with a non-reacting agent, myoglobin (mol. wt = 17000). It is postulated that the casein micelle has a sponge-like structure in which the 3 different subunits are distributed fairly uniformly. Micellar fractions obtained by differential centrifugation from skim-milk and colostrum were examined for their content of α s1 -, β-, κ- and para-κ-caseins by the use of CPA as described by Ribadeau Dumas (1968). The results confirm that there is a real difference in κ-casein content of the micelles, according to their rate of sedimentation.

Structure of the casein micelle. A proposed model

On the basis of complete permeability by high molecular weight reagents of casein micelles in milk and a uniform distribution of the 3 different casein subunits, a model of the micelle structure is proposed. It is composed of an average repeating unit of 1 κ-, 2 α s1 ;- and β-casein subunits assembled in a 3-dimensional network or branched polymer made of 130–130000 monomers, in which the trimers of κ-casein occupy the nodes and the copolymers of α s1 ;- and β-caseins make up the branches. All the associations between subunits are through non-covalent bonds. The chemical composition varies with the number of α s1 ;- and β;-casein subunits in the branches. This proposed structure is strongly supported by evidence from electron microscopy and a scale model has been made. It leads to an understanding of the role of κ-casein in micelle formation and opens new perspectives in explaining some properties of the caseins. It offers an interesting example of a new type of quaternary structure of protein subunits.

The cDNA-derived amino acid sequence of chick heat shock protein Mr 90,000 (HSP 90) reveals a "DNA like" structure: potential site of interaction with steroid receptors.

We report cDNA sequence, the complete derived aa sequence, and a predicted secondary structure of the chick hsp 90, a protein which has been found to form complexes with steroid hormone receptors. The modelling of the most negatively charged region A indicates that the alpha-helices of this portion of hsp 90 mimick DNA configuration. We propose that this region can, in absence of hormone, interact with and cap the positively charged DNA-binding domain of steroid receptors.

Amino acid composition of β-casein genetic variants

Abstract The presence of β-casein genetic variants has been demonstrated by Aschaffenburg (1961) . In order of decreasing mobility in urea starch-gel electrophoresis at pH 8.6, these variants have been termed β-casein A, B and C with the relative mobilities of 0.80, 0.76 and 0.70. In the present paper the amino acid composition of these variants is reported. The analyses were performed as independent and cooperative studies at both Dairy Institutes.

Etude de la constitution de la caseine de vache par chromatographie sur colonnes de diethylaminoethyl-cellulose en milieu uree

Abstract The fractionation of whole cow casein by DEAE-cellulose chromatography in urea, part of which was published in a preliminary note, has been continued. 1. 1. The composition of whole casein, determined by this method is the following: Minor fractions having an electrical charge lower than β-casein at pH 7, 13%; β-casein, 30%; κ-casein, 11%; αs-casein (minor fraction, 6%; αs1,2 38%). 2. 2. The quantitatively more important Fractions F10 and F7 (corresponding approximately to αs- and β-caseins) have been more specially studied. Fraction F7 contains only a minor impurity. It has been possible to eliminate this impurity by a method which will be published later, but Fraction F10 still contains some minor impurities.

Peptide mapping of intersubunit and receptor interactions of human choriogonadotropin

Seven peptides covering the entire sequence of human choriogonadotropin (hCG) alpha-subunit, eight peptides covering the hCG beta-subunit sequence and two peptides, one of human beta-lutropin and one of beta-thyrotropin were synthesized. We checked their ability to prevent reassociation between hCG alpha- and beta-subunits and between hCG and its receptor. Only the alpha 1-22, alpha 59-92 and beta 1-16 peptides inhibited the reassociation between the alpha- and beta-subunits of hCG with an ED50 of respectively 2 mM, 2 mM and 4 mM. Using porcine Leydig cells in primary culture, we showed that alpha 33-59, alpha 41-59 and beta 1-16 peptides decreased both the specific binding to the cell surface and the internalization of [125I]hCG and [125I]porcine LH with ED50 of 0.3, 0.1 and 0.5 mM, respectively. From these results, the following minimal area may be assigned, (i) to the alpha-beta interaction: alpha 5-16, alpha 52-72 (or alpha 59-70) and beta 8-16, and (ii) to the hormone-receptor association: alpha 41-45 and beta 8-16.

The protein structure code: what is its present status?

Current methods of prediction of protein conformation are reviewed and the algorithms on which they rely are presented. For non-homologous proteins and after cross-validation the reported methods exhibit a probability index, i.e. the per cent of correctly predicted residues per predicted residues, of 63-65% with a standard deviation of the order of 7% for three conformational states--helix, beta-strand and coil. This present limitation in the accuracy of predictions that use only the information of the local sequence can be related essentially to the effect of long-range interactions specific for each protein family. The methods based on sequence similarity can improve the accuracy of prediction by expressing explicitly the homology of the protein to be predicted with proteins in the database. In these circumstances the probability index can reach 87% with a standard deviation of 6.6%. This property can be used for modeling homologous proteins by aiding in amino acid sequence alignments. The prediction of the tertiary structure of a protein is still limited to the case of modeling a structure based on the known three-dimensional structure of a homologous protein.