Assessing the stability of a mutant protein withGROMACS

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GROMACS version: 2021.4-Ubuntu-2021.4-2
GROMACS modification: No

Hi,

So I am trying to assess the stability of a mutant protein with respect to its native conformation, in essence obtain a ddg value as well as possibly a break down of the components that contribute to the change in stability.
I have gone through the free energy of solvation tutorial, the lysozome tutorial, as well as the umbrella sampling tutorial. A superior recommended I use umbrella sampling but I don’t understand how the pulling concept would be applied to my single chain protein.

It might be asking for a lot but if someone who perhaps has experience in obtaining ddg values from gromacs or have done similar simulations could assist in directing me to a tutorial or protocol that may provide me with some guidance.

I did want to make use of thermodynamic integration for the MD simulations, however I was unable to find a tutorial to guide this process.

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Indeed, alchemical simulations (TI) are a better option than US for estimating the effects of mutation on a relative free energy between conformations.

You’ll have to have a model for the reference (here: unfolded) state, and it can be just a tiny peptide with the same transition, but you need to close the thermodynamic cycle to have a meaningful estimate.

Here’s a great paper that explains the key concept and choices: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8388617/

The PMX webserver will help you create alchemical amino acids: http://pmx.mpibpc.mpg.de/

In Fig 8 of that paper you will find the most popular strategies for running alchemical simulations. Depending on your choice, you will find different tools and tutorials; I should soon have a tutorial for case (D), I have been postponing it for a while, but there are excellent ones for a similar case (solvation free energy):

http://www.mdtutorials.com/gmx/free_energy/index.html
https://tutorials.gromacs.org/free-energy-of-solvation.html