Frédéric Cadet

Protein Block Expert (PBE): a web-based protein structure analysis server using a structural alphabet

Encoding protein 3D structures into 1D string using short structural prototypes or structural alphabets opens a new front for structure comparison and analysis. Using the well-documented 16 motifs of Protein Blocks (PBs) as structural alphabet, we have developed a methodology to compare protein structures that are encoded as sequences of PBs by aligning them using dynamic programming which uses a substitution matrix for PBs. This methodology is implemented in the applications available in Protein Block Expert (PBE) server. PBE addresses common issues in the field of protein structure analysis such as comparison of proteins structures and identification of protein structures in structural databanks that resemble a given structure. PBE-T provides facility to transform any PDB file into sequences of PBs. PBE-ALIGNc performs comparison of two protein structures based on the alignment of their corresponding PB sequences. PBE-ALIGNm is a facility for mining SCOP database for similar structures based on the alignment of PBs. Besides, PBE provides an interface to a database (PBE-SAdb) of preprocessed PB sequences from SCOP culled at 95% and of all-against-all pairwise PB alignments at family and superfamily levels. PBE server is freely available at .

Quantitative Determination of Sugar Cane Sucrose by Multidimensional Statistical Analysis of their Mid-Infrared Attenuated Total Reflectance Spectra

A fast and accurate method for determining the sucrose content of sugar cane juice has been developed. The application of principal component regression (PCR) has been proposed for the development of a prediction equation of sucrose content by mid-infrared spectroscopy. An attenuated total reflectance (ATR) cell is used in place of the more familiar transmission cell. PCR involves two steps: (1) the creation of new synthetic variables by principal component analysis (PCA) of spectral data, and (2) multiple linear regression (MLR) with these new variables. Results obtained by this procedure have been compared with those obtained by the conventional application of polarization.

Inhibition of palmito (Acanthophoenix rubra) polyphenol oxidase by carboxylic acids

Abstract The inhibition of palmito (Acanthophoenix rubra) polyphenol oxidase (PPO) is reported. Recently, two forms of palmito PPO were partially purified by hydrophobic chromatography. Inhibitory effects of various carboxylic acids on these two forms have been studied. Both forms showed identical behaviour towards the inhibitors studied. Cinnamic acid was found to have the greatest inhibitory effect (Ki = 0·06 mM). When the inhibitory effects of acids from the benzoic acid family and from the cinnamic acid family were compared, it was found that acids which possess a double bond between the benzene ring and the carboxylic function showed the highest inhibitory effect. This inhibitory effect was decreased by substitutions on the benzene ring. The influence of pH on the inhibitory effect of carboxylic acids on palmito PPO has also been investigated. Ki decreased with a decrease in pH. This effect is due to the fact that it is only the undissociated form (AH) of carboxylic acids that is responsible for inhibition of PPO. Inhibition constants (Ki for the AH form have been recalculated and were found to remain constant in the pH range studied (for benzoic acid Ki = 0·14 mM, for cinnamic acid Ki = 0·019 mM, for sorbic acid Ki = 0·15 mM).

Analysis on conservation of disulphide bonds and their structural features in homologous protein domain families

BackgroundDisulphide bridges are well known to play key roles in stability, folding and functions of proteins. Introduction or deletion of disulphides by site-directed mutagenesis have produced varying effects on stability and folding depending upon the protein and location of disulphide in the 3-D structure. Given the lack of complete understanding it is worthwhile to learn from an analysis of extent of conservation of disulphides in homologous proteins. We have also addressed the question of what structural interactions replaces a disulphide in a homologue in another homologue.ResultsUsing a dataset involving 34,752 pairwise comparisons of homologous protein domains corresponding to 300 protein domain families of known 3-D structures, we provide a comprehensive analysis of extent of conservation of disulphide bridges and their structural features. We report that only 54% of all the disulphide bonds compared between the homologous pairs are conserved, even if, a small fraction of the non-conserved disulphides do include cytoplasmic proteins. Also, only about one fourth of the distinct disulphides are conserved in all the members in protein families. We note that while conservation of disulphide is common in many families, disulphide bond mutations are quite prevalent. Interestingly, we note that there is no clear relationship between sequence identity between two homologous proteins and disulphide bond conservation. Our analysis on structural features at the sites where cysteines forming disulphide in one homologue are replaced by non-Cys residues show that the elimination of a disulphide in a homologue need not always result in stabilizing interactions between equivalent residues.ConclusionWe observe that in the homologous proteins, disulphide bonds are conserved only to a modest extent. Very interestingly, we note that extent of conservation of disulphide in homologous proteins is unrelated to the overall sequence identity between homologues. The non-conserved disulphides are often associated with variable structural features that were recruited to be associated with differentiation or specialisation of protein function.

Measurement of sugar content by multidimensional analysis and mid-infrared spectroscopy

The advent of more and more powerful micro-computers has allowed the introduction of multidimensional analysis in research laboratories. Complex mathematical treatments are now possible within a few seconds. Prediction equations that linked sucrose, fructose, glucose, total sugars and reducing sugars concentrations to the spectral data, were established by regression on the principal components. Very high correlation coefficient values between the first ten axes and the chemical values were obtained. The bias and standard deviation (S.D.) values obtained between reference and predicted values were good. From such aqueous biological samples containing a ternary mixture of sucrose, fructose and glucose it was possible to (i) identify the characteristic IR bands of these different sugars (and their combination: reducing sugars, total sugars)-using spectral pattern; and (ii) to specifically measure their concentrations with good accuracy.

Simultaneous Determination of Sugars by Multivariate Analysis Applied to Mid-Infrared Spectra of Biological Samples

We have investigated the use of principal component analysis (PCA) to describe and assess mid-infrared spectral data obtained from complex biological samples containing sucrose, fructose, and glucose. The correlation coefficients between spectral data and chemical values of each variable (sucrose, glucose, fructose, total sugars, and reducing sugars) showed that in each case, axes 1, 3, 4, and 5 had the highest values. These values also indicated which axes each variable was mostly correlated with. The results also showed that the samples were distributed according to their sucrose concentrations (or total sugars) along a concentration gradient in the projection plan formed between axes 1 and 3. No clear discrimination according to concentration was observed with other factorial maps. Prediction equations that linked sucrose, fructose, glucose, total sugar, and reducing sugars concentrations to the spectral data were established by regression on the principal component. Very high correlation coefficients values between the first 10 axes and the chemical values were obtained (between 0.9757 and 0.998). From such aqueous biological samples containing a ternary mixture of sucrose, fructose, and glucose, it was possible to (1) identify the characteristic IR bands of these different sugars (and their combination: reducing sugars/total sugars) and (2) to specifically measure their concentrations with a relatively good accuracy.

A kinetic study of the inhibition of palmito polyphenol oxidase by L-cysteine

Abstract Polyphenol oxidase (PPO) is responsible for browning reactions in fruits and vegetables. In order to inhibit these reactions during processing, the use of chemical inhibitors including thiols such as L-cysteine has been proposed. The effect of this thiol on PPO activity was studied in order to establish if it reacts with quinone and/or directly inhibits the enzyme. The inhibition of palmito PPO (catecholase activity, with 4-methylcatechol as substrate) by L-cysteine was characterized spectrophotometrically. The effect of increasing cysteine concentration with respect to incubation time was measured by polarography and the kinetic parameters calculated. Kinetic analysis using spectrophotometric assays showed a lag period suggesting that there is no quinone accumulation in the reaction mixture. The increase of absorbance at 300 nm observed with u.v.—vis spectra indicated that a colourless compound (thiol—quinone complex) was produced. The plots of log remaining enzyme activity vs incubation time were characterized by two straight lines suggesting two possible inactivation models: (i) the deactivation of one enzyme that proceeds in two steps or (ii) the existence of two isoenzymes that behave differently. We have shown that the inactivation process of PPO by cysteine followed this model: N → X[C] → I where N represents one native form, X represents an intermediate form, the structure of which depends on the cysteine concentration [C], and where I is the completely inactive form of the enzyme. These results showed that there is a direct irreversible inhibition of PPO by cysteine according to a two-step model. The analytical approach illustrated by the present study of palmito PPO inhibition by L-cysteine may be applied to other investigations of mechanisms of enzyme inhibition.

Enzyme kinetics by mid-infrared spectroscopy: β-fructosidase study by a one-step assay

An alternate method for enzyme study is proposed. Multidimensional statistical analysis applied on mid-infrared attenuated total reflectance spectra (Cadet et al. (1991) Appl. Spectrosc. 42, 166-172) collected during a kinetic allows a direct and fast quantification of the remaining substrate, as well as a one step enzymatic assay. Furthermore, the combination of these techniques may be used as a structural tool. The method applied to the study of beta-fructosidase is developed in this paper as an example. With appropriate calibration, the method may be extend to any enzyme.

Characterisation of the phosphoenolpyruvate carboxylase gene family in sugarcane (Saccharum spp.)

Abstract Phosphoenolpyruvate carboxylases (PEPCs) are encoded by a small multigenic family. In order to characterise this gene family in sugarcane, seven DNA fragments displaying a high homology with grass PEPC genes were isolated using polymerase chain reaction-based cloning. A phylogenetic study revealed the existence of four main PEPC gene lineages in grasses and particularly in sugarcane. Moreover, this analysis suggests that grass C4 PEPC has likely derived from a root pre-existing isoform in an ancestral species. Using the Northern-dot-blot method, we studied the expression of the four PEPC gene classes in sugarcane cv. R570. We confirmed that transcript accumulation of the C4 PEPC gene (ppc-C4) mainly occurs in the green leaves and is light-induced. We also showed that another member of this gene family (ppc-aR) is more highly transcribed in the roots. The constitutive expression for a previously characterised gene (ppc-aL2) was confirmed. Lastly, the transcript accumulation of the fourth PEPC gene class (ppc-aL1) was not revealed. Length polymorphism in non-coding regions for three PEPC gene lineages enabled us to develop sequence-tagged site PEPC markers in sugarcane. We analysed the segregation of PEPC fragments in self-pollinated progenies of cv. R570 and found co-segregating fragments for two PEPC gene lineages. This supports the hypothesis that diversification of the PEPC genes involved duplications, probably in tandem.

ANALYSIS OF NEAR-INFRARED SPECTRA OF SOME CARBOHYDRATES

Abstract Infrared, in particular near-infrared (NIR) is a very useful technique for the identification and the characterization of biomolecules. The complexity of the spectra tends to make direct interpretation difficult. However, from the spectroscopic representation of the factorial axes obtained by principal component analysis (PCA) performed on different sugar molecules, it is possible to determine the characteristic wavelengths (NIR) of monosaccharides, oligosaccharides and polysaccharides. The most characteristic wavelengths for monosaccharides are found to be: 1457 nm, 2062 nm, 2263 nm and 2440 nm; and for oligo- and polysaccharides: 1432 nm, 1931 nm, 2170 MI, 2310 nm and 2477 nm. These wavelengths, which have been identified from the spectral profiles, are the most appropriate for a quantitative study of unknown carbohydrate mixtures.