MamC from Magnetococcus marinus MC-1 has been shown to control the size of magnetite crystals in in vitro experiments, thereby demonstrating its potential as a candidate protein for the production of magnetite nanoparticles possibly useful in medical and other applications. However, the importance of the structure and aggregation state of the protein on the resulting biomimetic nanoparticles has not yet been assessed. One method normally used to prevent the aggregation of integral membrane proteins is the introduction of detergents during protein purification. In this study, results from protein aggregation following the addition of Triton-X100, DDM, and LDAO are presented. Magnetite particles formed in the presence of MamC purified using these three detergents were compared. Our results show that detergents alter the structure of the folded recombinant protein, thus preventing the ability of MamC to control the size of magnetite crystals formed chemically in vitro. Furthermore, we show that the introduction of detergents only at the dialysis process during the protein purification prevents its aggregation and allows for correct, functional folding of MamC. These results also indicate that the population of the active protein particles present at a certain oligomeric state needs to be considered, rather than only the oligomeric state, in order to interpret the ability of magnetosome recombinant proteins to control the size and/or morphology of magnetite crystals formed chemically in vitro.

MamC, an integral protein of the magnetosome membrane, has recently been proposed as a strong candidate to produce biomimetic (magnetosome-like) magnetite nanoparticles that could be used as an alternative to magnetosomes in different applications such as nanocarriers. The secondary structure of the protein contains two helical transmembrane domains connected by an α-helical loop oriented toward the magnetosome lumen. In this loop, the residues Glu66 and Asp70 seem to be responsible for a template effect that controls the nucleation and/or growth of biomimetic nanoparticles in vitro. In the present study, we have introduced a double mutation, E66A and D70A, in the sequence of MamC while working, for the first time, with the full-length protein. Our results show that this double mutation does not affect either the conformation or the stability of MamC, but it indeed makes the protein lose its functionality in terms of controlling the process of magnetite biomineralization in vitro. The present study shows that the ionotropic effect is not enough to account for the effect of the wild type MamC on the formation of BMNPs, but the template effect seems to rule such a process. Also, it shows that no other region of MamC is involved in controlling the process of magnetite biomineralization. Moreover, the stability of MamC in solution is only marginal, probably due to the absence of contacts established with the membrane lipid bilayer.

Magnetite nanoparticles in the magnetosomes formed by magnetotactic bacteria present unique magnetic properties that make them the ideal nanoparticle with potential use in several biotechnological applications. These magnetoliposomes are organelles formed by crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer. However, scaling up the production of these nanoparticles cannot be achieved currently because of the slow growth and the strict physiological characteristics of the magnetotactic bacteria. An alternative to solve this problem is the biomimetic approach, which involves the in vitro production of magnetite nanoparticles mediated by magnetosome membrane proteins, expressed as recombinant. MamC-mediated magnetite nanoparticles (BMNPs) have been recently proposed as some of the best biomimetic (magnetosome-like) nanoparticles for potential use in targeted drug delivery and hyperthermia treatments. However, the production of these crystals still needs to be scaled up. One of the critical steps in this scaling-up process is to find a method to keep the protein used as a template, MamC, fully functional over time, so that large amounts of protein could be produced at once. Since it has been previously demonstrated that the function of MamC in producing nanoparticles with optimal conditions is strictly linked to its structure, much care needs to be paid to ensure that such a structure is not affected by the protein preservation method. In the present study, MamC was produced and preserved under different conditions (lyophilization, cryoconservation, and refrigeration) for different time intervals. The efficiency of each preservation treatment was evaluated by studying the MamC conformation and oligomerization state and by analyzing the biomimetic crystals formed in the presence of this specific protein. Among all the methodologies assayed, only cryopreservation was able to keep the correct MamC structure and oligomerization state and, therefore, its activity.

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