Protein assembly for the design of new materials: characterization of interactions at amino-acid level between alpha-lactalbumin and lysozyme
Résumé
The final structure and stability of supramolecular objects result from interactions between proteins at amino-acid level. Their characterization is the basis for the design of new proteinbased biomaterials. In this study we focus on the interactions and assembly of bovine alphalactalbumin (LAC) with hen egg white lysozyme (LYS). While LYS interacts with the apo and holo forms of LAC (Ca2+ depleted and loaded form respectively), these interactions lead to the formation of micro-spheres with apoLAC only. Our objectives are to identify the amino-acids involved in the interactions and to establish the mechanism of such self-assembly
into suprastructures. The early step of the process is most probably the formation of heterodimers.
A structural characterization of both holoLAC-LYS and apoLAC-LYS dimers was performed in order to understand why micro-spheres are formed with apoLAC only. The surfaces of interaction of holoLAC with LYS and LYS with the holo and apo forms of LAC were determined using (1H, 15N) Nuclear Magnetic Resonance. Interacting amino-acids were identified according to the chemical shift perturbations measured during titration of one 15Nlabelled protein by its non-labelled partner. The titration of 15N-holoLAC by LYS indicates
that holoLAC interacts with LYS through its beta-domain (beta-sheet and flexible loop). The reverse titration indicates that LYS interacts with holoLAC mainly through its alpha-domain, including amino-acids of helices B and D and C- and N-terminal regions. Few residues of its beta-domain are also involved. In both cases the surfaces of interaction are localized on one side of the proteins and are composed of contiguous surface patches. LYS interacts with the apo form of LAC mainly through its alpha-domain and this surface patch is larger than with the holo form. This difference could be explained by the increased flexibility of the apoLAC
which is in its molten globule state at the working temperature (45°C). In order to know how proteins interact on an atom-to-atom basis, docking experiments were performed using the HADDOCK program. The interacting amino-acids were transformed into ambiguous intermolecular distance restraints to calculate hetero-dimers structures. This characterization is a prerequisite for the use of LAC and LYS micro-spheres as biomaterials, such as bioactive compound carrier. These findings give key elements to understand protein interaction and self-assembly process in mixed systems