## 3 Results and Discussion
Therefore, the protein backbone energetics are comparable in both Syn solutions and the result is the formation of a simply less strained protein backbone, when Syn solutions are more concentrated, experimenting higher shear stress and, consequently, becoming more aggregated. We further decided to perform dynamic light scattering (DLS) analysis of Syn solutions in order to verify the actual state of the Syn solutions subjected to centrifugation using different membrane centrifugal filters with different cut-off. In Figure 4A, the normalized volume distributions obtained by DLS for Syn solutions subjected to centrifugation using membrane centrifugal filters with 200 nm, 100 kDa, 50 kDa and 30 kDa cut-off as a function of the particle diameter (nm) are shown. From this figure it can be seen that the monomeric Syn protein populates the solutions and also that the normalized volume distribution is displaced towards lower sizes when decreasing the membrane centrifugal filters cut-off. This latter phenomenon has been observed in protein samples that can display interparticle and particle-solvent interactions that can cause substantial distortion in the apparent diffusion coefficients determined by DLS and hence affect the calculated hydrodynamic size [20]. These inter-protein and protein-solvent interactions are electrostatic interactions, sometimes referred to as virial effects [20]. The reduction in the particle size has been typically observed for high protein concentrations. Therefore, this means that when lowering the membrane centrifugal filters cut-off, by an increase in interactions among Syn protein monomers, leads to an increase in established intermolecular interactions (Figure 4A). In Figure 4B the volume peak area determined as a function of the membrane centrifugal filters pore size (nm) is shown [21]. From this figure, lowering the amount of the available Syn monomer species can be inferred when reducing the membrane centrifugal filters cut-off. In other words, when reducing the membrane centrifugal filters cut-off there is stress-induced in Syn protein solutions that can favor the Syn monomer species association, being also responsible for reducing the amount of the available Syn monomer species in solution (Figure 4B). Nevertheless, it should also be emphasized that accordingly to DLS measurements, we estimate the occurrence of a very low aggregation for the Syn protein and, therefore, Syn aggregated species numbers are <0.01% of the Syn species total population (mostly protein monomers). Overall, for Syn solutions, in 10 mM tris-HCl (pH 7), a decrease in the formation of intramolecular interactions and an increase in the formation of intermolecular interactions when reducing the membrane centrifugal filters pore size, under centrifugation conditions (Figure 3E-G), has been proposed. The centrifugation of NAYA solutions (in 10 mM tris-HCl, pH 7) did not seem to alter the type of the interactions formed. It deserves mentioning that the paradigm that emerges from most studies is that shear in the fluid mechanical sense is unlikely to damage most proteins by itself and that interfacial phenomena are critically important. Nevertheless, in the case of Syn, it is very likely that distortion of Syn species results, while the protein solutions go through the membrane centrifugal filters pores. Concerning the abovementioned interfacial phenomena, Syn is a disordered protein that is mostly exposed to the solvent and that can readily interact in the hydrophobic air/liquid interface, under agitation or under shear stress-imputed conditions such as those occurring during centrifugation. In our study, the gas phase of the Syn solutions was maintained constant by 50%, under centrifugation conditions. Additionally, the centrifugation parameters were roughly the same for the centrifuged protein solutions. Meaning, we are not expecting other phenomenon, besides Syn species distortion in the membrane centrifugal filters pores, to occur during centrifugation.