Understanding Conformational Changes in Human Serum Albumin and Its Interactions with Gold Nanorods: Do Flexible Regions Play a Role in Corona Formation?

The importance of protein-nanoparticle (NP) conjugates for biomedical applications has seen an exponential growth in the past few years. The protein corona formation on NPs with human serum albumin (HSA), being the most abundant protein in blood serum, has become one of the most studied protein analyses under NP-protein interactions as HSA is readily adsorbed on the surface of the NPs. Understanding the fate of the NPs in physiological media along with the change in biological responses due to the formation of the protein corona thus becomes important. We analyzed the HSA protein corona formation on gold nanorods (AuNRs) through different spectroscopic studies in addition to the effects of change in the protein concentration on the protein-NP interactions. Different imaging techniques such as high-resolution transmission electron microscopy, field emission scanning electron microscopy, and atomic force microscopy were used to determine the morphology and the dimensions of the nanorods and the protein-nanorod conjugates. Fourier-transform infrared data showed a reduction in the α-helix content and an increase in β-sheet content for the HSA-AuNR conjugate compared to the native protein. A decrease in steady-state fluorescence intensity occurred with instant addition of AuNR to HSA showing better and efficient quenching of Trp fluorescence for the lower concentration of protein. Time-correlated single photon counting results showed greater energy transfer efficiency and faster decay rate for higher concentrations of proteins. The circular dichroism study gives insight into the secondary structural changes due to unfolding, and a greater change was observed for lower concentrations of protein due to a thermodynamically stable protein corona formation. Surface-enhanced Raman spectroscopy (SERS) indicated the presence of aromatic residues such as Phe, Tyr, and Cys that appear to be close to the surface of the AuNRs in addition to hydrophobic interactions between AuNR and the protein. The disordered and flexible regions mapped onto HSA (PDB: 1AO6), predicted by the intrinsically disordered region predictors, point toward the interactions of similar residues with the nanorods observed from SERS and fluorescence studies. These studies could provide a clearer understanding of the interactions between HSA and AuNRs for possible biological applications.