Said Bouhallab

Charge and size drive spontaneous self-assembly of oppositely charged globular proteins into microspheres.

Controlled interactions and assembly of proteins with one another promise to be a powerful approach for generating novel supramolecular architectures. In this study, we report on the ability of oppositely charged globular proteins to self-assemble into well-defined micrometer-sized spherical particles under specific physicochemical conditions. We show that microspheres were spontaneously formed in all binary protein mixtures tested once the physicochemical conditions were optimized. The optimal pH value, initial protein concentrations needed to form microspheres, and protein stoichiometry in these microspheres varied and depended on the structural features of the mixed proteins. We show that charge compensation is required but not sufficient to guide optimal protein assembly and organization into microspheres. Size difference between protein couples (acidic and basic) is a key element that defines optimal pH value for microsphere formation and the protein molar ratio in the formed microspheres. Our findings give new elements that can help to predict the assembly behavior of various proteins in mixed systems.

Dynamic and supramolecular organisation of α-lactalbumin/lysozyme microspheres: A microscopic study

Apo alpha-lactalbumin (apo alpha-LA) and lysozyme (LYS), two homologous globular proteins have been shown to be able to interact and self-assemble to form microspheres. We report on the organisation and the mechanism of such protein assembly process using a variety of microscopic techniques. We demonstrated that proteins involved into apo alpha-LA/LYS microspheres exchange with those free in solution. The exchange process takes place from the periphery to the centre of the microspheres. The formed spherical particles observed after fixed incubation time were found to be either individual or aggregated according to the total protein concentration leading to structures with different size and morphology. It appears that protein assembly occurs throughout successive steps of aggregated spherical particles that reorganise into biggest isolated microspheres. Direct microscopic observations over time confirm that microspheres resulted from a reorganisation of aggregated, clustered nanospheres. We propose that the formation of apo alpha-LA/LYS microspheres follows an aggregation-reorganisation mechanism.

Kinetics and Structure during Self-Assembly of Oppositely Charged Proteins in Aqueous Solution

Self-assembly in aqueous solution of two oppositely charged globular proteins, hen egg white lysozyme (LYS) and bovine calcium-depleted α-lactalbumin (apo α-LA), was investigated at pH 7.5. The aggregation rate of equimolar mixtures of the two proteins was determined using static and dynamic light scattering as a function of the ionic strength (15-70 mM) and protein concentration (0.28-2.8 g/L) at 25 and 45 °C. The morphology of formed supramolecular structures was observed by confocal laser scanning microscopy. When the two proteins are mixed, small aggregates were formed rapidly that subsequently grew by collision and fusion. The aggregation process led on larger length scales to irregularly shaped flocs at 25 °C, but to monodisperse homogeneous spheres at 45 °C. Both the initial rate of aggregation and the fraction of proteins that associated decreased strongly with decreasing protein concentration or increasing ionic strength but was independent of the temperature.

Heteroprotein complex coacervation: A generic process.

Proteins exhibit a rich diversity of functional, physico-chemical and biodegradable properties which makes them appealing for various applications in the food and non-food sectors. Such properties are attributed to their ability to interact and assemble into a diversity of supramolecular structures. The present review addresses the updated research progress in the recent field of complex coacervation made from mixtures of oppositely charged proteins (i.e. heteroprotein systems). First, we describe briefly the main proteins used for heteroprotein coacervation. Then, through some selected examples, we illustrate the particularity and specificity of each heteroprotein system and the requirements that drive optimal assembly into coacervates. Finally, possible and promising applications of heteroprotein coacervates are mentioned.

Binding of Folic Acid Induces Specific Self-Aggregation of Lactoferrin: Thermodynamic Characterization.

In the study presented here, we investigated the interaction at pH 5.5 between folic acid (FA) and lactoferrin (LF), a positively charged protein. We found a binding constant Ka of 10(5) M(-1) and a high stoichiometry of 10 mol of FA/mol of LF. The size and charge of the complexes formed evolved during titration experiments. Increasing the ionic strength to 50 mM completely abolished the isothermal titration calorimetry (ITC) signal, suggesting the predominance of electrostatic interactions in the exothermic binding obtained. We developed a theoretical model that explains the complex triphasic ITC profile. Our results revealed a two-step mechanism: FA/LF interaction followed by self-association of the complexes thus formed. We suggest that 10 FA molecules bind to LF to form saturated reactive complexes (FA10/LF) that further self-associate into aggregates with a finite size of around 15 nm. There is thus a critical saturation degree of the protein, above which the self-association can take place. We present here the first results that provide comprehensive details of the thermodynamics of FA/LF complexation-association. Given the high stoichiometry, allowing a load of 55 mg of FA/g of LF, we suggest that FA/LF aggregates would be an effective vehicle for FA in fortified drinks.

Structure and Dynamics of Heteroprotein Coacervates.

Under specific conditions, mixing two oppositely charged proteins induces liquid-liquid phase separation. The denser phase, or coacervate phase, can be potentially applied as a system to protect or encapsulate different bioactive molecules with a broad range of food and/or medical applications. The optimization of the design and efficiency of such systems requires a precise understanding of the structure and the equilibrium of the nanocomplexes formed within the coacervate. Here, we report on the nanocomplexes and the dynamics of the coacervates formed by two well-known, oppositely charged proteins β-lactoglobulin (β-LG, pI ≈ 5.2) and lactoferrin (LF, pI ≈ 8.5). Fluorescence recovery after photobleaching (FRAP) and solid-state nuclear magnetic resonance (NMR) experiments indicate the coexistence of several nanocomplexes as the primary units for the coacervation. To our knowledge, this is the first evidence of the occurrence of an equilibrium between quite unstable nanocomplexes in the coacervate phase. Combined with in silico docking experiments, these data support the fact that coacervation in the present heteroprotein system depends not only on the structural composition of the coacervates but also on the association rates of the proteins forming the nanocomplexes.

Kinetics of the formation of β-casein/tannin mixed micelles

The complexation kinetics of β-casein with tannins were investigated by means of stopped flow and small-angle X-ray scattering (SAXS). Several small plant tannins have been considered: epigallocatechin-gallate (EGCG) from green tea and a set of oligomeric tannins from apples. We show that the kinetics are composed of two processes. The first process is a rapid uptake of tannins by the β-casein micelles over 40–100 ms and the second process is a slow reorganization of the tannin-dressed proteins into stable heavier micelles over a period of up to 200 s. In the first process, the protein segments in the cores of the micelles are rapidly coated by tannins. Detailed analysis of the SAXS profiles during the slow dynamics reveals that the system remains composed of micelles whose structural attributes evolve smoothly toward equilibrium values. The quantity of the bound tannins remains constant during the whole slow evolution of the system. We conclude that the dominant elementary events that drive the slow kinetics are the exchange processes of tannin-dressed proteins from one micelle to another.

Polar lipid composition of bioactive dairy co-products buttermilk and butterserum: Emphasis on sphingolipid and ceramide isoforms

Bioactive lipids of the milk fat globule membrane become concentrated in two co-products of the butter industry, buttermilk and butterserum. Their lipid composition is detailed here with special emphasis on sphingolipid composition of nutritional interest, determined using GC, HPLC and tandem mass spectrometry. Butterserum was 2.5 times more concentrated in total fat than buttermilk, with 7.7±1.5vs 19.5±2.9wt% and even more concentrated in polar lipids, with 1.4±0.2vs 8.5±1.1wt%. Both ingredients constitute concentrated sources of sphingomyelin (3.4-21mg/g dry matter) and contained low amounts of bioactive ceramides in a ratio to sphingomyelin of 1:5mol% in buttermilk and 1:10mol% in butterserum. Compared to other natural lecithins, these two co-products are rich in long and saturated fatty acids (C22:0-C24:0), contain cholesterol and could have interesting applications in neonatal nutrition, but also as brain-protective, hepatoprotective and cholesterol lowering ingredients.

How the presence of a small molecule affects the complex coacervation between lactoferrin and β-lactoglobulin

Heteroprotein complex coacervation corresponds to the formation of two liquid phases in equilibrium induced by the interaction of two oppositely charged proteins. The more concentrated phase known as coacervate phase, has attracted interest from several fields of science due to its potential applications for example for encapsulation and delivery of bioactives. Prior such application, it is necessary to understand how the presence of small ligands affects the complex coacervation. In this work, we report on the interaction of small ligand with individual proteins β-lactoglobulin (β-LG) and lactoferrin (LF) and consequences on their complex coacervation. ANS (8-Anilinonaphthalene-1-sulfonic acid), a fluorescent probe, was used as model ligand. While ANS did not interact with β-LG, it presented two sets of binding sites with LF inducing its self-aggregation. Depending on its concentration, ANS modulated the shape of β-LG-LF macromolecular assembly. Coacervates were observed for ANS/LF molar ratio <25 against amorphous aggregates for higher ANS/LF molar ratios. A maximum loading capacity of around 40mg of ANS per gram of LF in the formed heteroprotein coacervates was reached.

pH- and ionic strength-dependent interaction between cyanidin-3-O-glucoside and sodium caseinate

Understanding the mechanism of interaction between food proteins and bioactives constitutes the preliminary step to design food grade nanocarriers. We investigated the interaction between cyanidin-3-O-glucoside (C3G), and 20nm-sized sodium caseinate nanoparticles (NaCas) at pH 7 and pH 2 by fluorescence spectroscopy and dynamic light scattering. The characterization of the C3G-NaCas interaction indicated that the fluorescence quenching mechanism was predominantly static. C3G interacted with two sets of binding sites with association constants Ka of 106 and 105M-1. Electrostatic interactions dominated at pH 7, while hydrophobic effects were the main force at pH 2. Interestingly, the two sets of binding sites were discriminated by ionic strength at pH 7. The binding of C3G slightly modified the average diameter of NaCas nanoparticles without alteration of its surface charge suggesting a complexation of C3G molecules in the internal casein structure. Thus, NaCas constitutes a putative nanocarrier for anthocyanins in new functional foods.