RESUMEN
The purpose of this study was to investigate whether Human Serum Albumin (HSA) can bind N-methylated analogs of hot spots of native insulin. Three N-methylated derivatives of the A13-A19 fragment of native insulin were used: L(N-Me)YQLENY (1), LYQ(N-Me)LENY (2), and L(N-Me)YQ(N-Me)LENY (3). The studied N-methylated insulin fragments possess inhibiting potential against hormone aggregation. A variety of research techniques, including spectroscopic methods and microscopy assays, were used to study the interaction of HSA with the N-methylated insulin fragments. Based on spectroscopic measurements with Congo Red and Thioflavin T, all the analyzed N-methylated peptides were able to interact with the HSA surface. The CD spectrum registered for HSA in the presence of L(N-Me)YQLENY showed the smallest content of α-helix conformation, indicating the most compact HSA structure. Based on the results of MST, the dissociation constants (Kd) for complexes of HSA and peptides 1-3 were 19.2 nM (complex 1), 15.6 nM (complex 2), and 8.07 nM (complex 3). Microscopy assays, dynamic light scattering measurements as well as computer simulation of protein-ligand interaction also confirmed the possibility of docking the N-methylated inhibitors within HSA.