Jean-François Lefèvre

Stabilization of proteins by glycosylation examined by NMR analysis of a fucosylated proteinase inhibitor.

Here we investigate the effects of the naturally occuring threonine-linked L-fucose moiety on the structure, dynamics and stability of the proteinase inhibitor PMP-C (Pars intercerebralis major peptide C). The three-dimensional structure of PMP-C fucosylated on Thr 9 has been determined by NMR spectroscopy and simulated annealing. The f ucose ring is very well ordered, held in place by hydrophobic and hydrogen bond interactions with Thr 16 and Arg 18. Comparing the NMR data and the structure of the fucosylated inhibitor with those of the nonf ucosylated form shows that conformational changes only occur in the vicinity of the fucose moiety. Nevertheless, a comparative analysis of the exchange rates of amide protons indicates that fucosylation is responsible for an overall decrease of the dynamic fluctuations of the molecule. This correlates well with an increase in stability of ∼1 kcal mol−1 as monitored by thermal denaturation.

Characterization of Antibacterial COOH-terminal Proenkephalin-A-derived Peptides (PEAP) in Infectious Fluids IMPORTANCE OF ENKELYTIN, THE ANTIBACTERIAL PEAP209–237 SECRETED BY STIMULATED CHROMAFFIN CELLS

Proenkephalin-A (PEA) and its derived peptides (PEAP) have been described in neural, neuroendocrine tissues and immune cells. The processing of PEA has been extensively studied in the adrenal medulla chromaffin cell showing that maturation starts with the removal of the carboxyl-terminal PEAP209–239. In 1995, our laboratory has shown that antibacterial activity is present within the intragranular chromaffin granule matrix and in the extracellular medium following exocytosis. More recently, we have identified an intragranular peptide, named enkelytin, corresponding to the bisphosphorylated PEAP209–237, that inhibits the growth of Micrococcus luteus (Goumon, Y., Strub, J. M., Moniatte, M., Nullans, G., Poteur, L., Hubert, P., Van Dorsselaer, A., Aunis, D., and Metz-Boutigue, M. H. (1996) Eur. J. Biochem. 235, 516–525). As a continuation of this previous study, in order to characterize the biological function of antibacterial PEAP, we have here examined whether this COOH-terminal fragment is released from stimulated chromaffin cells and whether it could be detected in wound fluids and in polymorphonuclear secretions following cell stimulation. The antibacterial spectrum shows that enkelytin is active against several Gram-positive bacteria including Staphylococcus aureus, but it is unable to inhibit the Gram-negative bacteria growth. In order to relate the antibacterial activity of enkelytin with structural features, various synthetic enkelytin-derived peptides were tested. We also propose a computer model of synthetic PEAP209–237 deduced from 1H NMR analysis, in order to relate the antibacterial activity of enkelytin with the three-dimensional structure. Finally, we report the high phylogenetic conservation of the COOH-terminal PEAP, which implies some important biological function and we discuss the putative importance of enkelytin in the defensive processes.

The reconstruction of the relaxation matrix from an incomplete set of nuclear overhauser effects

Abstract Internal motions in a macromolecule and indirect magnetization transfers (spin diffusion) prevent simple interpretation of NOE values in terms of structural and dynamic parameters in the course of the study of a macromolecule. A method for separating these two problems by first analyzing the NOES with respect to the relaxation rate constants is proposed. NOE values for 1 D or 2D experiments are obtained as nonlinear functions of the ϱ and σ parameters by solving the Bloch equations. A general least-squares minimization routine that makes use of the Hessian matrix of second derivatives was found to be efficient in determining iteratively the set of parameters that statistically correlates the corresponding calculated NOEs to a set of experimental NOES. The procedure was applied to probe the internal mobility of the deoxyriboses in a self-complementary hexamer, d (CGTACG) 2 . It was found that H 2′ -H 2″ and H 1′ -H 2″ -vectors are subject to internal motions whose amplitudes vary as we move along the sequence.

Fluorimetric study of the complex between yeast phenylalanyl-tRNA synthetase and tRNA-Phe. 2. Evidence for an asymmetric behaviour of the enzyme.

The variations of several spectroscopic properties of yeast tRNA-Phe and phenylalanyl-tRNA synthetase upon complex formation, were used to study the stoichiometry of the complex in different experimental conditions. In all cases, for the tRNA-Phe-enzyme complex, in the absence of other ligands, the saturations of the different conformational changes monitored for both macromolecules, are achieved at a 2:1 tRNA/enzyme stoichiometry. Phenylalanine does not modify this saturation. In contrast, the presence of 1 mM ATP induces an asymmetric behaviour of the synthetase: two tRNAs are still bound per enzyme molecule but the conformational change of the latter is completed upon binding of a single tRNA molecule.

Computing the geometry of a molecule in dihedral angle space using n.m.r.-derived constraints: A new algorithm based on optimal filtering

We have developed a method based on optimal filtering to determine the three-dimensional structure of a protein from n.m.r.-derived constraints, using the dihedral angle internal representation of the molecule. It differs from currently proposed methods in that it directly produces estimates of errors on the parameters that are refined, hence providing an image of the minimum that has been found. A similar algorithm had already been proposed using cartesian co-ordinates as independent parameters, encoded in PROTEAN2. We found that using dihedral angles significantly reduces the computational burden of the technique, and provides better control over a priori informations that can be used, such as geometric restrictions for proline residues and informations from vicinal coupling constants. Performance of the method, encoded in FILMAN, is demonstrated by application to the folding of a ten-residue alanine polypeptide, to the geometric cyclization of an 11-residue peptide, as well as on the folding of a medium size protein, i.e. tendamistat. The validity of the error estimates on the dihedral angles produced by FILMAN is discussed.

Kinetic studies of the modulation of ada promoter activity by upstream elements.

We have determined the kinetics of initiation of transcription of the wild‐type ada promoter by abortive initiation assays. In the absence of activation, it is a weak promoter, with an association constant KB and an isomerization rate constant k2 comparable to those obtained under the same conditions for other positively regulated promoters (0.36 x 10(7) M‐1 and 1.7 x 10(‐2) s‐1, respectively, at 37 degrees C and 50 mM NaCl, on a supercoiled template). As already observed for other promoters, these constants are modulated by varying the supercoiling of the plasmid. However, the strength of the promoter (given by the KB.k2 product) remains almost constant, because the maximum value of KB and k2 are obtained for different values of the superhelical density. The ada promoter has a stretch of seven adenosine residues (A7) in its upstream region. We have analysed the effect of this upstream sequence on the efficiency of initiation of the ada promoter by comparing the wild‐type sequence with an up‐mutant promoter characterized by the inversion of the central base pair in the sequence (A7) leading to the sequence (A3TA3). Although the mutation, which is located outside the promoter consensus regions, has no effect on the isomerization step, it affects the equilibrium constant KB that characterizes the association step. In the mutant promoters, the supercoiling of the plasmid modulates the isomerization and association constants in such a way that both KB and k2 are maximum for the same superhelical density (‐0.05), leading to a 12‐fold increase of the strength of the promoter, on a supercoiled template.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and dynamic studies of two antigenic loops from haemagglutinin: A relaxation matrix approach

SummaryWe have investigated the dynamics and structural behaviour of two antigenic peptides using 1H NMR. The two cyclic peptides mimic the antigenic site A of influenza haemagglutinin protein; they only differ in the way they were cyclized and in the size of their respective linkers. Homonuclear relaxation parameters extracted from a complete NOE matrix were interpreted in terms of local dynamics. A set of distance constraints was deduced from these parameters which allowed 3D models to be constructed using distance geometry. NOE back-calculation was used to check the validity of the final models. Strong variations of internal motion amplitude have been found in both peptides along their backbone. Motions with high amplitudes have been localized in the Gly-Pro-Gly sequence which forms a β-turn in both structures.

The Dynamics of Oligonucleotides and Peptides Determined by Proton NMR

It is widely accepted that knowledge of the structure of a biological macromolecule is an important key to understanding its biological function. On the other hand, it is known that these macromolecules have adaptable structures that are specifically modified during their interactions with their partners. This adaptability is generally due to the flexibility of structural motifs in the macromolecule that are arranged in a specific way in space. The flexibility is possible because the potential energy around the average structure of the motif is quite flat, so that the structural motif is continuously moving between several isoenergetical structures, giving rise to the so called internal motion of the macromolecule. The knowledge of this internal motion is an important step toward understanding the biological function of a macromolecule and, consequently, toward engineering macromolecules.

The Investigation of Protein Dynamics Via the Spectral Density Function

Spectral density mapping provides direct access to information on protein dynamics, by the solution of the equations for heteronuclear relaxation rates, with no assumptions as to the nature of the molecule or its dynamic behaviour. Reduced spectral density mapping allows the detailed characterisation of a protein’s motions at a lower experimental burden than the full approach. Spectral density profiles may readily be interpreted to yield information on flexibility or slow conformational exchange and to describe the anisotropic nature of the molecule.

Fast abortive initiation of uvrA promoter in a supercoiled plasmid studied by stopped-flow techniques

In order to follow the fast kinetics of abortive initiation (lag time from 1 ms to 10 s), we have built a stopped‐flow apparatus equipped for fluorescence detection. The small volume used for each assay (35 μl), and the short dead time (∼0.5 ms) are the essential advantages of this apparatus. Supercoiling of DNA affects considerably the initiation of transcription from the uvrA promoter. It decreases the lag time due to the isomerisation process 3‐fold. Nevertheless, it does not change significantly the product K B k 2, which is indicative of promoter strength and shows that uvrA is an ‘association‐limited’ promoter. The presence of the LexA repressor increases the lag time considerably. At least for small RNA polymerase concentrations this increase is stronger for supercoiled than for linearized DNA.