Effect of Gold Nanoparticles on the Conformation of Bovine Serum Albumin: Insights from CD Spectroscopic Analysis and Molecular Dynamics Simulations

Abstract

With the development of nanotechnology, there is growing interest in using nanoparticles (NPs) for biomedical applications, such as diagnostics, drug delivery, imaging, and nanomedicine. The protein's structural stability plays a pivotal role in its functionality, and any alteration in this structure can have significant implications, including disease progression. Herein, we performed a combined experimental and computational study of the effect of gold NPs with a diameter of 5 nm (5 nm Au-NPs) on the structural stability of bovine serum albumin (BSA) protein in the absence and presence of NaCl salt. Circular dichroism spectroscopy showed a loss in the secondary structure of BSA due to the synergistic effect of Au-NPs and NaCl, and Thioflavin T fluorescence assays showed suppressed β-sheet formation in the presence of Au-NPs in PBS, emphasizing the intricate interplay between NPs and physiological conditions. Additionally, molecular dynamics (MD) simulations revealed that 5 nm Au-NP induced changes in the secondary structure of the BSA monomer in the presence of NaCl, highlighting the initial binding mechanism between BSA and Au-NP. Furthermore, MD simulations explored the effect of smaller Au-NP (3 nm) and nanocluster (Au-NC with the size of 1 nm) on the binding sites of the BSA monomer. Although the formation of stable BSA-Au conjugates was revealed in the presence of NPs of different sizes, no specific protein binding sites were observed. Moreover, due to its small size, 1 nm Au-NC decreased helical content and hydrogen bonds in the BSA monomer, promoting protein unfolding more significantly. In summary, this combined experimental and computational study provides comprehensive insights into the interactions among Au nanosized substances, BSA, and physiological conditions that are essential for developing tailored nanomaterials with enhanced biocompatibility and efficacy.

Figure 1

CD spectroscopy of the samples investigated in this study with the enlarged region around 201–260 nm.

Figure 2

ThT fluorescence measurement results recorded within 120 min from the start of stirring: · BSA with no Au-NPs; · BSA with 10 nM Au-NPs (d = 5 nm); -·- polynomial fit for the “BSA with no Au-NPs”; - - - polynomial fit for the “BSA with 10 nM Au-NPs (d = 5 nm)”.

Figure 3

(A) Secondary structure of BSA monomer observed at the last 25 ns of the simulations. (B) Time evolution of H-bonds in Domain I in the absence and presence of Au-NP (d = 5 nm), averaged among different runs.

Figure 4

Time evolution of the intermolecular distances (nm) between the center of masses of (A) BSA monomer and 1 nm Au-NC, (C) BSA monomer and 3 nm Au-NP, and (B) BSA monomer and 5 nm Au-NP. Three runs of the simulated systems with NaCl and three runs of the simulated systems with no NaCl are shown separately for each system.

Figure 5

Time-evolution of the distances between the COM of the Au-NS and BSA monomer subdomains in the absence of NaCl in (A) run 1 (1 nm Au-NC), (B) run 2 (1 nm Au-NC), (C) run 3 (1 nm Au-NC), (D) run 1 (3 nm Au-NP), (E) run 2 (3 nm Au-NP), (F) run 3 (3 nm Au-NP), (G) run 1 (5 nm Au-NP), (H) run 2 (5 nm Au-NP), and (I) run 3 (5 nm Au-NP).

Figure 6

Time-evolution of the distances between the COM of the Au-NS and BSA monomer subdomains in the presence of 0.15 M NaCl in A) run 1 (1 nm Au-NC), B) run 2 (1 nm Au-NC), C) run 3 (1 nm Au-NC), D) run 1 (3 nm Au-NP), E) run 2 (3 nm Au-NP), F) run 3 (3 nm Au-NP), G) run 1 (5 nm Au-NP), H) run 2 (5 nm Au-NP), I) run 3 (5 nm Au-NP).

Figure 7

Representative snapshots of the simulated systems at the end of the simulations depicted from a single 50 ns run (yellow: Au; red: Na⁺ ions within 1 nm around the protein; green: Cl^– ions within 1 nm around the protein; secondary structure of the BSA protein: blue, red, violet: helices; yellow and cyan: unstructured bend and turn, D-I: Domain I, D-II: Domain II, D-III: Domain III; water molecules are not shown for clarity): (A) BSA monomer in the presence of NaCl, (B) BSA monomer and 1 nm Au-NC in the presence of NaCl, (C) BSA monomer and 3 nm Au-NP in the presence of NaCl, (D) BSA monomer and 5 nm Au-NP in the presence of NaCl, (E) BSA monomer in the absence of NaCl, (F) BSA monomer and 1 nm Au-NC in the absence of NaCl, (G) BSA monomer and 3 nm Au-NP in the absence of NaCl, (H) BSA monomer and 5 nm Au-NP in the absence of NaCl.