Bernard Roux

Role of metal ions on the secondary and quaternary structure of alkaline phosphatase from bovine intestinal mucosa

Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa (BIAP) is an homodimeric metalloenzyme, containing one Mg2+ and two Zn2+ ions in each active site. ApoBIAP, prepared using ion‐chelating agents, exhibited a dramatic decrease of its hydrolase activity, concomittant to conformational changes in its quaternary structure. By rate‐zonal centrifugation and electrophoresis, we demonstrated, for the first time, that the loss of divalent ions leads to some monomerization process for a metal‐depleted alkaline phosphatase. Divalent ions are also involved in the secondary and tertiary structures. Metal‐depletion induced more exposure of some Trp residues and hydrophobic regions to the solvent (as proved by intrinsic and ANS fluorescences). These changes might correspond to the disappearance of α‐helices and/or turns with a concomittant appearance of unordered structures and β‐sheets (as probed by FTIR spectroscopy). For BIAP, three steps of temperature‐induced changes were exhibited, while for apoBIAP, only one step was exhibited at 55°C. Our work on BIAP showed two main differences with alkaline phosphatase from Escherichia coli. The loss of the divalent ions induces protein monomerization and the total recovery of enzyme activity by divalent ion addition to apoBIAP was not obtained. Proteins 1999;37:310–318. ©1999 Wiley‐Liss, Inc.

ANTHEPROT: a package for protein sequence analysis using a microcomputer.

A simple microcomputer package is described to make the theoretical analysis of protein sequences. Several methods designed to compare two sequences, to model proteolytic reactions and to predict the secondary structure, the hydrophobic/hydrophilic regions and the potential antigenic sites of proteins have been included in an Apple II microcomputer software. The package comprises 21 programs as well as the secondary structure database of Kabsch and Sander (1983).

Behavior of a GPI-anchored protein in phospholipid monolayers at the air–water interface

The interaction between alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein (AP-GPI), and phospholipids was monitored using Langmuir isotherms and PM-IRRAS spectroscopy. AP-GPI was injected under C16 phospholipid monolayers with either a neutral polar head (1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC)) or an anionic polar head (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS)). The increase in molecular area due to the injection of protein depended on the surface pressure and the type of phospholipid. At all surface pressures, it was highest in the case of DPPS monolayers. The surface elasticity coefficient E, determined from the pi-A diagrams, allowed to deduct that the AP-GPI-phospholipid mixtures presented a molecular arrangement less condensed than the corresponding pure phospholipid films. PM-IRRAS spectra suggested different protein-lipid interactions as a function of the nature of the lipids. AP-GPI modified the organization of the DPPS deuterated chains whereas AP-GPI affected only the polar group of DPPC at low surface pressure (8 mN/m). Different protein hydration layers between the DPPC and DPPS monolayers were suggested to explain these results. PM-IRRAS spectra of AP-GPI in the presence of lipids showed a shape similar to those collected for pure AP-GPI, indicating a similar orientation of AP-GPI in the presence or absence of phospholipids, where the active sites of the enzyme are turned outside of the membrane.

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme.

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts. By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.

Role of histones H1 and H3 in the maintenance of chromatin in a compact conformation. Study with an immobilized enzyme.

Chromatin polynucleosomes have been digested with trypsin immobilized on collagen membranes. This method allows the mild removal of the most accessible histone fragments simply by dipping the enzymatic membrane into the chromatin solution, without modification of its ionic and chemical composition. These results demonstrate that the removal of H1 does not affect the higher‐order structure of chromatin and that only the elimination of the terminal regions of H3 leads to the unfolding of H1‐depleted fibres. This observation suggests that structural changes reported in many previous works were not due to only the removal of H1 but to a concomitant unbinding from DNA of the N‐terminal domain of H3.

Study of chromatin organization with trypsin immobilized on collagen membranes.

Trypsin immobilized on collagen membranes has been used to digest chromatin polynucleosomes. With this method, the use of protease inhibitor is avoided and the digestion time easily controlled simply by taking the membrane out of the chromatin solution. Its most fundamental advantage is however to allow the mild removing of the most accessible histone fragments without addition of salt then without perturbation of their ionic environment. Degradation of histone fractions were correlated with conformational changes using circular dichroism and electric birefringence measurements. On digestion, the sign of birefringence reversed, becoming negative, and an increase of molar ellipticity was observed. These changes reflecting the unfolding of DNA correspond to the digestion of Hl and also of fragments of H3. This would indicate that H3 and particularly its basic terminal regions, play a fundamental role in the maintenance of chromatin in a compact structure.

Penetration of a GPI-anchored protein into phospholipid monolayers spread at the air/water interface

Abstract Mammalian alkaline phosphatases (AP) belong to glycosylphosphatidyl inositol (GPI) anchored proteins family, which are localised and clustered on the outer layer of the plasma membranes forming microdomains. Using Langmuir film and polarisation modulation infrared reflection absorption spectroscopy (PMIRRAS) techniques, the penetration process of the protein into a phospholipid monolayer have been studied at the air–buffer interface. The penetration of AP-GPI in distearoylphosphatidylcholine monolayers (DSPC) induces a more important surface pressure increase than in dioleoylphosphatidylcholine (DOPC) monolayer. However, the exclusion surface pressure rather similar for both lipids, 20.5 and 22 mN m−1 for, respectively, DSPC and DOPC, indicates that the AP-GPI cannot, in similar conditions, insert by itself into bilayer membranes of either biological or mimetic origin. PMIRRAS suggests that the pure acyl chains perdeuterated DSPC (d70-DSPC) interact with Mg2+ present into the buffer. AP-GPI inserts progressively into the d70-DSPC monolayer changing the environment of phospholipid molecules. Amide I band exhibits α helix and β-sheets components with a predominance of the α helix. The shapes, intensities and positions of the amide I and II bands suggest for the α helix an orientation perpendicular to the interface after a period of molecular reorganisation.

A FTIR spectroscopy evidence of the interactions between wheat germ agglutinin and N-acetylglucosamine residues

Wheat germ agglutinin (WGA), a lectin binding a N‐acetyl‐D‐neuraminic acid (NeuNAc) and/or N‐acetyl‐D‐glucosamine (GlcNAc) group, was studied by Fourier transform infrared (FTIR) spectroscopy. Deconvolution of the FTIR spectrum of WGA alone indicated the presence of few α‐helices and β‐sheets, in contrast to many other lectins. These results agree with previous WGA crystal data. The WGA conformational changes, induced by GlcNAc‐bearing liposomes or GlcNAc oligomers, were studied by infrared differential spectroscopy. The GlcNAc binding to WGA resulted in a decrease of turns and α‐helices and a concomitant appearance of β‐sheets, inducing more or less peptidic N‐H deuteration.

Study of a chromatin domain different from bulk chromatin in barley nuclei

Abstract A method for fractionation of barley chromatin was developed. A soluble fraction, eluted from micrococcal nuclease-digested nuclei, was recovered and studied in comparison with the low ionic strength soluble bulk chromatin obtained after lysis of nuclei. The eluted fraction contained mainly mononucleosomes with less than 10% dinucleosomes. It was highly micrococcal nuclease-sensitive, totally depleted in the highest molecular mass H1 subspecies, and enriched both in the lowest Mr proteins and in other acid-soluble proteins. The soluble bulk chromatin exhibited protein and DNA contents analogous to those of the low ionic strength insoluble fraction. In addition, high-mobility-group proteins purification was carried out. Four species were identified. They were almost exclusively localized in the highly micrococcal nuclease-sensitive fraction.

Very low field electric birefringence of high molecular weight flexible polyelectrolytes

Abstract The electric birefringence of a natural flexible polyelectrolyte (calf thymus DNA) is measured at different field strengths and concentrations with rectangular and reversing pulses, or with continuous sine waves. It is shown that reproducible and field-independent relaxation times can only be obtained by using very low electric fields and concentrations. Under these conditions, and not withstanding the polydispersity and deformability, a well-defined long relaxation mechanism is demonstrated. It is attributed to the orientation of the polyelectrolyte as a whole. The field dependence of the corresponding birefringence fraction is studied. A typical saturation curve is thus obtained, although the total birefringence does not saturate, even at high field values.