Effect of Substrate-Binding Domain of Fusion Protein on Binding with Poly(3-hydroxybutyrate)-Based Nanocarrier

High Resolution Image Download MS PowerPoint Slide The macromolecular stability of fused proteins is usually provided by punctual structural modifications along the primary sequence of the chimera. Among these, the disulfide bond formation stands out as a key parameter for enabling the generation of structurally constrained protein architectures that may prevent unfolding or aggregation of a biologically active conformation. Moreover, unpaired and reactive cysteine groups are prone to undergo thiol–disulfide interchange reactions and cause conformational and functional switch. In this study, we report on the design and analysis of fused proteins constituted by superfolder green fluorescent protein (sfGFP) fused with a substrate-binding domain (SBD) from Ralstonia pickettii that display the capacity of binding to poly(3-hydroxybutyrate) (PHB) interfaces of micro and nanocarriers. To verify the impact of the cysteine residues along the SBD sequence on the domain’s affinity toward PHB, we have genetically engineered distinct sequences characterized by the absence or substitution of cysteines. The interaction profile of each protein with PHB microparticles and nanoparticles was compared via surface-sensitive techniques.