Protein assembly for the design of new materials: characterization of interactions at amino acids levels between a-lactalbumin and lysozyme

The final structure and stability of supramolecular objects result from interactions between proteins at amino-acid level. Their characterizatio n is the basis for the design of new protein- based biomaterials. In this study we focus on the i nteractions and assembly of bovine alpha- lactalbumin (LAC) with hen egg white lysozyme (LYS) . While LYS interacts with the apo and holo forms of LAC (Ca 2+ depleted and loaded form respectively), these inte ractions lead to the formation of micro-spheres with apoLAC only. Our objectives are to identify the amino-acids involved in the interactions and to est ablish the mechanism of such self-assembly into suprastructures. The early step of the process is most probably the formation of hetero- dimers. A structural characterization of both holoL AC-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 ( 1 H, 15 N) Nuclear Magnetic Resonance. Interacting amino-ac ids were identified according to the chemical shift perturba tions measured during titration of one 15 N- labelled protein by its non-labelled partner. The t itration of 15 N-holoLAC by LYS indicates that holoLAC interacts with LYS through its beta-do main (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 su rfaces 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 b y 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 t ransformed into ambiguous intermolecular distance restraints to calculate het ero-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. in assembly for the design of new materials: characterization of interactions at amino acids levels between a-lactalbumin and lysozyme