Model

Model Section

In our project, molecular dynamics simulations, molecular docking, and quantum chemical calculations played a crucial role in optimizing the protein-probe system and exploring the structure of the probe molecules. We not only studied the interaction between mutated human serum albumin (HSA) and the CO-1080 probe through docking simulations but also analyzed the structure and energy levels of the probe molecules through quantum chemical calculations. These model studies provided important theoretical support for optimizing the bioimaging performance of the probe.

BTB Model: Molecular Docking of HSA and CO-1080

To investigate how site-directed mutations in HSA improve its binding affinity to the CO-1080 dye, we conducted molecular docking simulations. The focus was on evaluating the binding of wild-type HSA and several recombinant HSA mutants, particularly rHSA-C476G, with CO-1080. Theoretical simulations using the Glide docking mode predicted the binding interactions between wild-type HSA and rHSA-C476G with CO-1080. Mod_Fig 1A shows these simulation results, highlighting the significant differences in binding between the mutated protein (rHSA-C476G) and CO-1080 compared to the wild-type.

Mod_Fig 1: Simulation of wild-type HSA and rHSA-C476G binding to CO-1080

By comparing docking scores, Mod_Fig 1B presents the non-covalent binding interactions between CO-1080 and various HSA mutants. The results showed that rHSA-C476G had a significantly better docking score than wild-type HSA, indicating that the C476G mutation improved the binding affinity with CO-1080. Additionally, as shown in Mod_Fig 1C, the binding energy of rHSA-C476G with CO-1080 was significantly higher than that of other mutants, further proving that the enhanced binding was due to the breaking of the disulfide bond and the exposure of free –SH groups.

These findings reveal the key role of protein mutations in improving probe binding, particularly the Cys476 mutation, which increased the number of binding sites by breaking the disulfide bond, making it easier for CO-1080 to bind to the protein. The improvement in binding energy and docking scores provides a solid theoretical foundation for the enhanced NIR-II imaging performance of the probe.

BBB Model: Side-Chain Length and Molecular Stability of Probes

In addition to the CO-1080 probe, we extended our research by analyzing a series of Cn-1080 dyes with different side-chain lengths, evaluating their molecular conformation, stability, and optical performance. Using density functional theory (DFT) calculations, we optimized the geometries of the Cn-1080 probes and thoroughly examined the variations in HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) energy levels.

Mod_Fig 2: Schematic structure of Cn-1080 dyes

Mod_Fig 3A illustrates the optimal molecular conformations of Cn-1080 dyes with different side-chain lengths, showing how the length of the side chains affects the overall structure of the molecule. The changes in side-chain length not only influence the torsional amplitude of the probe but also play a key role in its stability within biological environments. Through molecular dynamics simulations, we further analyzed the torsional amplitude and variance of these molecules. Mod_Fig 3B and 3C respectively display the torsional amplitude and torsional angle variance of different probes, helping us understand the flexibility and structural stability of the probe molecules in biological systems.

Mod_Fig 3: Analysis of Cn-1080 dyes with different side-chain lengths

Additionally, through quantum chemical calculations, Mod_Fig 3E presents the HOMO and LUMO energy levels of different Cn-1080 dyes. These energy levels were calculated using Gaussian software (b31yp/6-31g(d)), providing critical insights for optimizing the optical performance of the probes. We found that the length of the side chains affected the electronic energy levels of the probes, which in turn influenced their fluorescence performance. By tuning these electronic structures, we were able to optimize the luminescent properties of the probes to achieve higher NIR-II imaging efficacy.

Conclusion

Through detailed molecular simulations and calculations of the BTB and BBB models, we have reached several key conclusions. Firstly, the C476G mutation in HSA significantly enhanced its binding efficiency with the CO-1080 probe, exhibiting better binding energy and docking scores, which translated to greater stability and imaging performance in NIR-II imaging. Secondly, the Cn-1080 probe molecules with varying side-chain lengths demonstrated excellent structural stability and optical performance. Through molecular dynamics simulations and quantum chemical calculations, we found that probes with optimal side-chain lengths achieved more stable molecular conformations and superior optical properties.

These results provide a theoretical foundation for further optimization of protein-probe complexes and offer technical support for future bioimaging applications, especially for NIR-II imaging in complex biological environments such as the BTB and BBB.