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Delphine Salvatore

Interactions at molecular scale and assembly mechanisms between two proteins: α-lactalbumin and lysozyme



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Published on 11 April 2011
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Thesis presented April 11, 2011

Abstract:
Many researches focus on the design of biomaterials using the self-assembly properties of proteins since the resulting supramolecular objects exhibit many potential applications for food, pharmaceutical and biotechnology industries. The understanding of the driving forces governing protein assembly is a prerequisite to control the stability, morphology and functionalities of the resulting objects. This study focuses on the binary system composed of oppositely charged proteins: the lysozyme (LYS) and the α-lactalbumin​ (LAC). LYS interacts with calcium-loaded (holo) and calcium-depleted (apo) forms of LAC to form heterodimers. Only the apoLAC-LYS dimers further assemble into supramolecular objects with morphologies depending on the temperature. When apoLAC is in a native conformation, the assembly results in amorphous aggregates, whereas ordered spherical objects, called microspheres, are obtained when it adopts a conformational state with a particular flexibility, called "molten globule". In order to understand how the information contained at molecular scale are spread on microscopic scale, my PhD thesis objectives were to characterize the interactions involved at molecular level and to establish the assembly mechanism. The identification of the residues composing the heterodimers interfaces evidenced a preferential orientation of proteins resulting from electrostatic attractions, and the formation of tetramers by association of the apoLAC-LYS dimers. Sub-micrometric particles growth, from the nuclei formed by the aggregation of tetramers, is independent of the temperature and governed by collision and fusion of smaller particles. At the final stage of the mechanism, the conformational state of apoLAC plays an important role. The coalescence of particles and reorganization of proteins into microspheres is favored by the flexibility of the molten globule and probably involve hydrophobic interactions. Finally, the microspheres formation does not involved drastic secondary structure changes of assembled proteins, which explains the​ reversibility of the objects and the dynamic behavior of assembled proteins. The experimental strategy adopted to perform this work can be translated to study the assembly of other protein binary systems and the obtained fundamental elements can be used to envisage potential applications of the microspheres.

Keywords:
α-lactalbumin, lysozyme, interaction surface, protein assembly, supramolecular objects

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