As a chemist, I cannot perform actual molecular dynamics simulations, but I can provide you with a general outline of how to approach this problem and what you might expect to observe.1. Choose a suitable molecular dynamics MD simulation software: There are several MD simulation software packages available, such as GROMACS, AMBER, NAMD, and CHARMM. Choose one that you are familiar with or that is most suitable for your specific protein-ligand system.2. Prepare the protein-ligand complex: Obtain the 3D structure of the protein-ligand complex, either from experimental data e.g., X-ray crystallography or NMR or by performing a docking simulation. Ensure that the complex is properly solvated and ionized, and that the force field parameters for both the protein and ligand are correctly assigned.3. Set up the simulation parameters: Define the simulation parameters, such as the time step, simulation length, and ensemble e.g., NVT or NPT . Choose a range of temperatures to study the effect of temperature on the binding affinity. For example, you could start with a low temperature e.g., 278 K and incrementally increase it to a high temperature e.g., 318 K .4. Perform the MD simulations: Run the MD simulations for each temperature. Make sure to equilibrate the system at each temperature before collecting data for analysis.5. Analyze the results: Calculate the binding energy and stability of the complex at each temperature using various analysis tools provided by the MD simulation software. Some common methods to estimate binding energy include the MM/PBSA Molecular Mechanics/Poisson-Boltzmann Surface Area and MM/GBSA Molecular Mechanics/Generalized Born Surface Area approaches. Additionally, monitor the root-mean-square deviation RMSD and root-mean-square fluctuation RMSF of the protein and ligand to assess the stability of the complex.6. Interpret the findings: Based on the binding energy and stability data, determine how temperature affects the binding affinity of the protein-ligand complex. Generally, as temperature increases, the protein and ligand may exhibit increased flexibility, leading to changes in the binding affinity. However, the specific effect of temperature on the binding affinity will depend on the nature of the protein-ligand interaction and the system being studied.7. Validate the results: To ensure the reliability of your findings, it is essential to validate the results using experimental techniques, such as isothermal titration calorimetry ITC or surface plasmon resonance SPR , which can provide direct measurements of binding affinity as a function of temperature.