Adsorption on montmorillonite prevents oligomerization of Bt Cry1Aa toxin.

The adsorption of the insecticidal Cry1Aa protein from Bacillus thuringiensis (Bt-toxin) on a model clay surface was studied to understand the structural changes of the protein induced by the clay surface. We studied the adsorption of the monomeric and soluble oligomeric forms of the Cry1Aa toxin as a function of pH and ionic strength conditions on montmorillonite, which is an electronegative phyllosilicate. Cry1Aa secondary structure was determined from the amide I' FTIR absorption profiles. Accessibility to the solvent was determined by NH/ND exchange to characterize conformational flexibility of the different states of the Cry1Aa protein. The size distribution of Cry1Aa solutions was obtained by dynamic light scattering (DLS). From combined DLS and FTIR measurements, we conclude that montmorillonite traps the Cry1Aa toxin in its monomeric state, preventing the oligomerization of the protein. The oligomeric forms were adsorbed onto the clay without significant structural changes.

Vesicle permeabilization by purified soluble oligomers of prion protein: a comparative study of the interaction of oligomers and monomers with lipid membranes.

The conversion of normal cellular prion protein (PrP) into its pathological isoform, scrapie PrP, may occur at the cell surface or, more probably, in late endosomes. The early events leading to the structural conversion of PrP appear to be related to the presence of more or less stable soluble oligomers, which might mediate neurotoxicity. In the current study, we investigate the interaction of alpha-rich PrP monomers and beta-rich size-exclusion-chromatography-purified PrP oligomers with lipid membranes. We compare their structural properties when associated with lipid bilayers and study their propensities to permeabilize the membrane at physiological pH. We also study the influence of the N-terminal flexible region (residues 24-103) by comparing full-length PrP(24-234) and N-terminally truncated PrP(104-234) oligomers. We showed that both 12-subunit oligomers cause an immediate and large increase in the permeability of the membrane, whereas equivalent amounts of monomeric forms cause no detectable leakage. Although the two monomeric PrP constructs undergo an alpha-to-beta conformational change when bound to the negatively charged membrane, only the full-length form of monomeric PrP has a weak fusogenic effect. Finally, the oligomers affect the integrity of the membrane differently from the monomers, independently of the presence of the N-terminal flexible domain. As for other forms of amyloidogenesis, a reasonable mechanism for the toxicity arising from PrP fibrillization must be associated with low-molecular-weight oligomeric intermediates, rather than with mature fibrils. Knowledge of the mechanism of action of these soluble oligomers would have a high impact on the development of novel therapeutic targets.

Kinetics of conformational changes of fibronectin adsorbed onto model surfaces.

Fibronectin (FN), a large glycoprotein found in body fluids and in the extracellular matrix, plays a key role in numerous cellular behaviours. We investigate FN adsorption onto hydrophilic bare silica and hydrophobic polystyrene (PS) surfaces using Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) in aqueous medium. Adsorption kinetics using different bulk concentrations of FN were followed for 2h and the surface density of adsorbed FN and its time-dependent conformational changes were determined. When adsorption occurs onto the hydrophilic surface, FN molecules keep their native conformation independent of the adsorption conditions, but the amount of adsorbed FN increases with time and the bulk concentration. Although the protein surface density is the same on the hydrophobic PS surface, this has a strong impact on the average conformation of the adsorbed FN layer. Indeed, interfacial hydration changes induced by adsorption onto the hydrophobic surface lead to a decrease in unhydrated beta-sheet content and cause an increase in hydrated beta-strand and hydrated random domain content of adsorbed FN. This conformational change is mainly dependent on the bulk concentration. Indeed, at low bulk concentrations, the secondary structures of adsorbed FN molecules undergo strong unfolding, allowing an extended and hydrated conformation of the protein. At high bulk concentrations, the molecular packing reduces the unfolding of the stereoregular structures of the FN molecules, preventing stronger spreading of the protein.

Study of uranyl sorption onto hematite by in situ attenuated total reflection-infrared spectroscopy.

In this paper, we present results of ATR-IR spectroscopy of uranyl complexes adsorbed on hematite. This method allowed the in situ recording of infrared spectra of uranyl sorbed on hematite in presence of aqueous solution and to detect one peak at 906 cm(-1) attributed to the antisymmetric O=U=O stretching. The intensity of the peak increases with pH, but its shape does not evolve, indicating that the same surface species is responsible for the sorption in the pH range 5-8. The reversibility experiments confirm that the hematite deposit reacts in the same way as dispersed suspensions. Measurement of the stretching frequency of nitrate ions coming from electrolyte showed a pure electrostatic adsorption and exclude the formation of a ternary complex with uranyl.

Fate of prions in soil: trapped conformation of full-length ovine prion protein induced by adsorption on clays.

Studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. We consider the surface-induced conformational changes that the recombinant ovine prion protein (ovPrP) may undergo under different pH conditions when interacting with soil minerals of highly adsorptive capacities such as montmorillonite. The conformational states of the full-length ovine prion protein adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pD range 3.5-9, using FTIR spectroscopy. The in vitro pH-induced conversion of the alpha-helical monomer of ovPrP into oligomers of beta-like structure prone to self-aggregation does not occur when the protein is adsorbed on the clay surface. The conformation of the trapped ovPrP molecules on montmorillonite is pH-independent and looks like that of the ovPrP solvated state at pD higher than 7, suggesting the major role of Arg and Lys residues in the electrostatic origin of adsorption. The uneven distribution of positively and negatively charged residues of the ovPrP protein would promote a favored orientation of the protein towards the clay, so that not only the basic residues embedded in the N-terminal flexible part but also external basic residues in the globular part of the protein might participate to the attractive interaction. From these results, it appears unlikely that the interaction of normal prions (PrP(C)) with soil clay surfaces could induce a change of conformation leading to the pathogenic form of prions (PrP(Sc)).

Structural effects of drying and rehydration for enzymes in soils: a kinetics-FTIR analysis of alpha-chymotrypsin adsorbed on montmorillonite.

The effects of desiccation and rehydration cycles encountered by extracellular enzymes in soils are studied on -chymotrypsin adsorbed on montmorillonite. The controlled hygrometric FTIR cell used in this study enables to monitor drying and rehydration steps undergone by the -chymotrypsin-montmorillonite suspension or by the enzyme alone. Relative humidity (RH) determines the amount of deuterated water in the FTIR cell atmosphere. The molar water/protein ratio (W/P) as well as the conformational and solvation states of the enzyme have been determined using H/D exchange monitored by FTIR-transmission spectroscopy. When the W/P ratio decreases from 3500 to approximately 400, unfolding of beta-secondary structure in three different domains involves about 8% of the polypeptide backbone with respect to the most solvated states. Desiccation induces beta-unfolding, which opens channels allowing free vapor water molecules to diffuse into the enzyme at 15% RH. On drying to 0% RH, displacements of internal water (H2O) in the enzyme are demonstrated by reverse peptide isotopic exchanges (COND ==> CONH). Specific beta-structures, only formed in highly solvated states, sequester around 20 internal H2O molecules. Indeed, most of the unfolded secondary structures during the drying step are refolded at W/P approximately 1000 during rehydration. However, self-association hinders the recovery of the initial closed tertiary structure. The pD-dependent structural changes controlling inward and outward water diffusion are suppressed, whether the protein is initially in an adsorbed state or in solution. Changes in secondary structures encountered during desiccation/rehydration cycle are similar for the protein either free or in the adsorbed state. Thus domains that are unfolded by adsorption are not concerned by the desiccation/rehydration cycle.

Conformation, orientation, and adsorption kinetics of dermaseptin B2 onto synthetic supports at aqueous/solid interface.

The antimicrobial activity of cationic amphipathic peptides is due mainly to the adsorption of peptides onto target membranes, which can be modulated by such physicochemical parameters as charge and hydrophobicity. We investigated the structure of dermaseptin B2 (Drs B2) at the aqueous/synthetic solid support interface and its adsorption kinetics using attenuated total reflection Fourier transform infrared spectroscopy and surface plasmon resonance. We determined the conformation and affinity of Drs B2 adsorbed onto negatively charged (silica or dextran) and hydrophobic supports. Synthetic supports of differing hydrophobicity were obtained by modifying silica or gold with omega-functionalized alkylsilanes (bromo, vinyl, phenyl, methyl) or alkylthiols. The peptide molecules adsorbed onto negatively charged supports mostly had a beta-type conformation. In contrast, a monolayer of Drs B2, mainly in the alpha-helical conformation, was adsorbed irreversibly onto the hydrophobic synthetic supports. The conformational changes during formation of the adsorbed monolayer were monitored by two-dimensional Fourier transform infrared spectroscopy correlation; they showed the influence of peptide-peptide interactions on alpha-helix folding on the most hydrophobic support. The orientation of the alpha-helical Drs B2 with respect to the hydrophobic support was determined by polarized attenuated total reflection; it was around 15 +/- 5 degrees. This orientation was confirmed and illustrated by a molecular dynamics study. These combined data demonstrate that specific chemical environments influence the structure of Drs B2, which could explain the many functions of antimicrobial peptides.

Conformational changes of enzymes adsorbed at liquid-solid interface: relevance to enzymatic activity.

FTIR with attenuated total reflectance spectroscopy was used to study in situ adsorption of enzymes at water-solid interfaces to better understand how conformational changes may monitor enzymatic activity. Because the adsorption process depends on hydrophobic and electrostatic interactions, conformational changes were studied as a function of the nature of the adsorbing substrates, which are hydrophobic or hydrophilic in character. The adsorption kinetics of two examples of serine enzymes, alpha-chymotrypsin (alpha-chym) and Humicola lanuginosa lipase (HLL), were studied. The secondary structure and solvation of the adsorbed enzymes were both compared to the dissolved enzymes. The positively charged alpha-chym was adsorbed on a negatively charged hydrophilic support with minor structural changes, but the negatively charged lipase had no affinity for a similar support. Both enzymes were strongly retained on the hydrophobic support. The secondary and tertiary structures of the alpha-chym adsorbed on the hydrophobic support were strongly altered, which correlates to the inhibition of enzymatic hydrolysis. The specific solvation obtained for the adsorbed HLL is consistent with the existence of the open conformer in relation to the enhanced enzymatic activity at the water-hydrophobic interface.

Structural changes of human serum albumin immobilized on chromatographic supports: a high-performance liquid chromatography and Fourier-transform infrared spectroscopy study.

Chiral stationary phases obtained by immobilization of HSA on [C8] and [C18] reversed-phases and on poly(1-vinylimidazole)-coated silica were tested to resolve DL-tryptophan, N-benzoyl-DL-phenylalanine, RS-oxazepam and RS-warfarin racemic mixtures. Parameters of enantioselectivity measured in HPLC are correlated to structural and solvation states for adsorbed HSA, evaluated by FTIR spectroscopy. HSA immobilized on [PVI]-anion-exchangers is highly selective. HSA molecules are not self-associated, only unfolded for a small hydrophobic helix. The HSA-coated reversed-phases have a lower selectivity. Unfolding is larger but the indole-benzodiazepine chiral site is preserved and remains accessible.

Detection of short-lived electrogenerated species by Raman microspectrometry

Raman microprobe spectrometry has been applied to the characterization of unstable species generated electrochemically at a microelectrode (radius in the 10 microm range). The ability of the spectroelectrochemical method to detect short-lived intermediates is directly related to its capability to probe small volumes. Raman microprobe spectrometry is appropriate for electrochemical applications because it allows the analysis of approximately 1 microm3 of solution. In spectroelectrochemical experiments, such a volume corresponds to a reaction layer of 1 microm thickness. Potentially, this technique can allow the observation of species with lifetimes of the order of 1 ms. To enhance the capabilities of this spectroscopic technique, we utilized it in combination with steady-state voltammetry at a microelectrode, to increase the concentration of unstable intermediates near the electrode surface. To determine the detection limit of this combined technique, we varied the base concentration as a means for varying the lifetime the radical cation electrogenerated from 9,10-dichloroanthracene. Well-resolved resonance Raman spectra were obtained for this radical cation when the lifetime was > or = 0.1 ms. This short time resolution achieved with micro-Raman spectroelectrochemistry makes this technique a powerful tool for the characterization of short-lived intermediates that are generated electrochemically in solution.

Conformational Changes of Bovine Serum Albumin Induced by Adsorption on Different Clay Surfaces: FTIR Analysis.

Interactions between proteins and clays perturb biological activity in ecosystems, particularly soil extracellular enzyme activity. The pH dependence of hydrophobic, hydrophilic, and electrostatic interactions on the adsorption of bovine serum albumin (BSA) is studied. BSA secondary structures and hydration are revealed from computation of the Amide I and II FTIR absorption profiles. The influence of ionization of Asp, Glu, and His side chains on the adsorption processes is deduced from correlation between p(2)H dependent carboxylic/carboxylate ratio and Amide band profiles. We quantify p(2)H dependent internal and external structural unfolding for BSA adsorbed on montmorillonite, which is an electronegative phyllosilicate. Adsorption on talc, a hydrophobic surface, is less denaturing. The results emphasize the importance of electrostatic interactions in both adsorption processes. In the first case, charged side chains directly influence BSA adsorption that generate the structural transition. In the second case, the forces that attract hydrophobic side chains toward the protein-clay interface are large enough to distort peripheral amphiphilic helical domains. The resulting local unfolding displaces enough internal ionized side chains to prevent them from establishing salt bridges as for BSA native structure in solution. On montmorillonite, a particular feature is a higher protonation of the Asp and Glu side chains of the adsorbed BSA than in solution, which decreases coulombic repulsion. Copyright 2000 Academic Press.

Chymotrypsin Adsorption on Montmorillonite: Enzymatic Activity and Kinetic FTIR Structural Analysis.

Soils have a large solid surface area and high adsorptive capacities. To determine if structural and solvation changes induced by adsorption on clays are related to changes in enzyme activity, alpha-chymotrypsin adsorbed on a phyllosilicate with an electronegative surface (montmorillonite) has been studied by transmission FTIR spectroscopy. A comparison of the pH-dependent structural changes for the solution and adsorbed states probes the electrostatic origin of the adsorption. In the pD range 4.5-10, adsorption only perturbs some peripheral domains of the protein compared to the solution. Secondary structure unfolding affects about 15-20 peptide units. Parts of these domains become hydrated and others entail some self-association. However, the inactivation of the catalytic activity of the adsorbed enzyme in the 5-7 pD range is due less to these structural changes than to steric hindrance when three essential imino/amino functions, located close to the entrance of the catalytic cavity (His-40 and -57 residues and Ala-149 end chain residue), are oriented toward the negatively charged mineral surface. When these functions lose their positive charge, the orientation of the adsorbed enzyme is changed and an activity similar to that in solution at equivalent pH is recovered. This result is of fundamental interest in all fields of research where enzymatic activity is monitored using reversible adsorption procedures. Copyright 1999 Academic Press.