Investigation at Residue Level of the Early Steps during the Assembly of Two Proteins into Supramolecular Objects

Abstract

Understanding the driving forces governing protein assembly requires the characterization of interactions at molecular level. We focus on two homologous oppositely charged proteins, lysozyme and α-lactalbumin, which can assemble into microspheres. The assembly early steps were characterized through the identification of interacting surfaces monitored at residue level by NMR chemical shift perturbations by titrating one (15)N-labeled protein with its unlabeled partner. While α-lactalbumin has a narrow interacting site, lysozyme has interacting sites scattered on a broad surface. The further assembly of these rather unspecific heterodimers into tetramers leads to the establishment of well-defined interaction sites. Within the tetramers, most of the electrostatic charge patches on the protein surfaces are shielded. Then, hydrophobic interactions, which are possible because α-lactalbumin is in a partially folded state, become preponderant, leading to the formation of larger oligomers. This approach will be particularly useful for rationalizing the design of protein assemblies as nanoscale devices.

Anisotropic Interactions in Protein Mixtures: Self Assembly and Phase Behavior in Aqueous Solution

“…7 Including the above square well potential, we obtained K d = 446 μM. Thus, the short-ranged attraction improves agreement with the experimental phase behavior as well as consistency at the two-protein level.…”

Anisotropic protein–protein interactions due to ion binding

“…Lysozyme distribution (brown iso-density surface) around ˛-lactalbumin (ghosted). The arrow shows the direction of the molecular dipole moment (pH 7) of the latter, while residues marked in red are those most perturbed upon binding as measured using NMR [31]. reversal for protein-protein interactions, which is indeed also observed experimentally [30].…”

“…4, top model. NMR experiments, where lysozyme is titrated into a solution of ˛-lac, later confirms [31] the anisotropic protein-protein interaction (also shown in Fig. 3).…”

Heteroprotein complex coacervation: A generic process

“…The detailed molecular mechanism was further elucidated through selective identification of the amino-acids involved on the interacting protein surfaces [13]. These experiments were performed by titration of one 15 Nlabelled protein with its unlabelled partner using NMR chemical shift perturbation measurements.…”

“…Another important requirement is protein stoichiometry in order to respect a charge and size compensation principle [12]. The extreme sensitivity to change in pH and ionic strength suggests the predominant role of the electrostatic interactions in the formation of the coacervate even if dipolar attraction and hydrophobic interactions are also cited as driving forces in heteroprotein coacervation [13]. In the presence of salt the coulombic interactions between the proteins and the entropic contribution due to counter-ions release are reduced.…”