Conformational Transitions upon Maturation Rule Surface and pH-Responsiveness of α-Lactalbumin Microparticulates.

De novo designed protein supramolecular structures are nowadays attracting much interest as highly performing biomaterials. While a clear advantage is provided by the intrinsic biocompatibility and biodegradability of protein and peptide building blocks, developing sustainable and green bottom up approaches for finely tuning the material properties still remains a challenge. Here, we present an experimental study on the formation of protein microparticles in the form of particulates from the protein α-lactalbumin using bulk mixing in water solution and high temperature. Once formed, the structure and stability of these spherical protein condensates change upon further thermal incubation while the size of aggregates does not significantly increase. Combining advanced microscopy and spectroscopy methods, we prove that this process, named maturation, is characterized by a gradual increase of amyloid-like structure in protein particulates, an enhancement in surface roughness and in molecular compactness, providing a higher stability and resistance of the structure in acidic environments. When dissolved at pH 2, early stage particulates disassemble into a homogeneous population of small oligomers, while the late stage particulates remain unaffected. Particulates at the intermediate stage of maturation partially disassemble into a heterogeneous population of fragments. Importantly, differently matured microparticles show different features when loading a model lipophilic molecule. Our findings suggest conformational transitions localized at the interface as a key step in the maturation of amyloid protein condensates, promoting this phenomenon as an intrinsic knob to tailor the properties of protein microparticles formed via bulk mixing in aqueous solution. This provides a simple and sustainable platform for the design and realization of protein microparticles for tailored applications.