supramolecular structures formed under the different physicochemical conditions
of the medium tested was performed.
Our research group had further extended these interaction studies using a
multiscale approach from molecular to microscopic level. The mechanism of
cross-assembly between oppositely charged proteins was investigated using various
mixtures of globular proteins derived from milk or egg white (Table 3). We thus
studied in depth the process of interaction–assembly between α-La from bovine
milk and egg-white LYS. The use of the α-La in its two conformational forms holo
(with calcium) and apo (without calcium) aimed to clarify the role of protein
conformation in the assembly process. At the molecular level, LYS interacts with
holo and apo forms of α-La at pH 7.5 to form oligomeric structures with a
dissociation constant in the micromolar range in both cases [135]. The affinity
constant between the two proteins decreases with increasing ionic strength,
reflecting the involvement of electrostatic interactions. However, only the apo
α-La/LYS mixture spontaneously assembles into supramolecular objects, leading
to liquid–liquid phase separation.
In the case of the α-La–LYS system, the cross-assembly mechanism between
these two proteins is controlled by both (a) the conformation of α-La and (b) the
molecular interaction with LYS, leading to the formation of oligomers of specific
conformations [135]. Characterisation of interactions at the molecular level was
further performed to understand the driving forces behind such protein assembly.
The early steps of assembly were characterised through the identification of the
amino acid involved in the interacting surfaces of the proteins. This was monitored
by NMR chemical shift perturbations by titrating one
15 N-labelled protein with its
unlabelled partner [136]. These authors showed that α-La has a narrow interaction
site on the formed heterodimers, whereas LYS exhibited interaction sites scattered
on a broader surface. Further assembly into tetramers requires additional interaction
sites between apo α-La–LYS heterodimers. The absence of bound calcium on apo
α-La exposes another negatively charged patch on the protein surface, which gives
a second site for interacting with LYS. Within the formed tetramers, most of the
electrostatic charge patches on the protein surface are shielded, enhancing the
contribution of the hydrophobic patches on the tetramer surface for further
assemblies. Again, the increased flexibility of apo α-La compared to holo α-La
favourably exposes some hydrophobic residues on the surface of tetramer. Then,
hydrophobic interactions are assumed to contribute to subsequent assembly
throughout the formation of larger oligomers of LYS and apo α-La. These experimental results agree and match well with Monte Carlo simulations that show
preferential alignment between proteins resulting from the strength of their dipole
moments [136].
Interestingly, the morphology of cross-assembled objects between apo α-La and
LYS was found to be temperature dependent: polydisperse amorphous aggregates
are obtained by mixing the two proteins at a temperature below 25
C compared
with well-ordered spherical particles when the mixing is carried out above 30
C [3].
It is well-established that the apo form of α-La changes its conformation above
27
C from “native-like” to a “molten globule” conformation that is more flexible.
88
S. Bouhallab and T. Croguennec
of the medium tested was performed.
Our research group had further extended these interaction studies using a
multiscale approach from molecular to microscopic level. The mechanism of
cross-assembly between oppositely charged proteins was investigated using various
mixtures of globular proteins derived from milk or egg white (Table 3). We thus
studied in depth the process of interaction–assembly between α-La from bovine
milk and egg-white LYS. The use of the α-La in its two conformational forms holo
(with calcium) and apo (without calcium) aimed to clarify the role of protein
conformation in the assembly process. At the molecular level, LYS interacts with
holo and apo forms of α-La at pH 7.5 to form oligomeric structures with a
dissociation constant in the micromolar range in both cases [135]. The affinity
constant between the two proteins decreases with increasing ionic strength,
reflecting the involvement of electrostatic interactions. However, only the apo
α-La/LYS mixture spontaneously assembles into supramolecular objects, leading
to liquid–liquid phase separation.
In the case of the α-La–LYS system, the cross-assembly mechanism between
these two proteins is controlled by both (a) the conformation of α-La and (b) the
molecular interaction with LYS, leading to the formation of oligomers of specific
conformations [135]. Characterisation of interactions at the molecular level was
further performed to understand the driving forces behind such protein assembly.
The early steps of assembly were characterised through the identification of the
amino acid involved in the interacting surfaces of the proteins. This was monitored
by NMR chemical shift perturbations by titrating one
15 N-labelled protein with its
unlabelled partner [136]. These authors showed that α-La has a narrow interaction
site on the formed heterodimers, whereas LYS exhibited interaction sites scattered
on a broader surface. Further assembly into tetramers requires additional interaction
sites between apo α-La–LYS heterodimers. The absence of bound calcium on apo
α-La exposes another negatively charged patch on the protein surface, which gives
a second site for interacting with LYS. Within the formed tetramers, most of the
electrostatic charge patches on the protein surface are shielded, enhancing the
contribution of the hydrophobic patches on the tetramer surface for further
assemblies. Again, the increased flexibility of apo α-La compared to holo α-La
favourably exposes some hydrophobic residues on the surface of tetramer. Then,
hydrophobic interactions are assumed to contribute to subsequent assembly
throughout the formation of larger oligomers of LYS and apo α-La. These experimental results agree and match well with Monte Carlo simulations that show
preferential alignment between proteins resulting from the strength of their dipole
moments [136].
Interestingly, the morphology of cross-assembled objects between apo α-La and
LYS was found to be temperature dependent: polydisperse amorphous aggregates
are obtained by mixing the two proteins at a temperature below 25
C compared
with well-ordered spherical particles when the mixing is carried out above 30
C [3].
It is well-established that the apo form of α-La changes its conformation above
27
C from “native-like” to a “molten globule” conformation that is more flexible.
88
S. Bouhallab and T. Croguennec