Protein-DNA Binding Affinity

User discussions
1

GROMACS Version: gromacs/gromacs (Docker image) 2022.2
GROMACS Modifications: No

I use GROMACS to calculate the binding free energy between a protein (FHD) and DNA.

To estimate the free energy of binding, I used the following formula:

ΔG_binding=G_complex−G_protein−G_DNA

I calculated:

  • Potential energy (enthalpy approximation) using:
    gmx energy -f md_0_10.edr -o enthalpy.xvg
  • Entropy using principal component analysis:
    1. gmx covar -s md_0_10.tpr -f md_0_10_fit.xtc -o eigenval.xvg -v eigenvec.trr -av average.pdb
    2. gmx anaeig -v eigenvec.trr -entropy yes

However, some computed binding free energies (ΔG) are positive, which suggests unfavorable binding — contradicting biological expectations.

My Questions:

  • Is this approach valid for estimating absolute binding free energies from MD simulations?
  • Why are my free energies positive?
  • Are there common sources of error or limitations in this method?
  • Should I consider only relative ΔG (ΔΔG) instead of absolute values?

I would appreciate any advice or recommended best practices for estimating protein–DNA binding affinities using GROMACS.