Dear Daeseong Kim , to the best of my knowledge. The stability of a protein evaluated with FoldX is expressed as a positive value due to the way the software calculates and represents folding energies. Here’s a detailed explanation of why this is the case and how it relates to protein stability:

1. FoldX Energy Calculations

Folding Energy Representation: In protein folding studies, the Gibbs free energy change (ΔG) is typically used to assess stability. A negative ΔG indicates that a protein is more stable in its folded state compared to its unfolded state. However, FoldX may present these values in a way that can be confusing, often leading to positive values for ΔG. Here, a positive ΔΔG indicates that the mutation decreases stability.

Positive Values Indicate Unfavorable Stability: When FoldX reports a positive ΔG value, it suggests that the protein is less stable. For example, if ΔG (WT) = 30 kcal/mol and ΔG (Mut) = 40 kcal/mol, this indicates that the wild-type protein is more stable than the mutant.

The calculation for ΔΔG would be:

ΔΔG=ΔG(Mut)−ΔG(WT)=40−30=10 kcal/mol

This negative value indicates that the mutation stabilizes the protein.

Negative ΔΔG Indicates Greater Stability: If ΔΔG is negative, it means that the mutant protein is more stable than the wild type. Thus, in your example, if you have:ΔG (WT) = 30 kcal/mol ΔG (Mut) = 20 kcal/mol Then: ΔΔG=20−30=−10 kcal molΔΔG=20−30=−10 kcal mol

2. Comparison with Schrödinger Software

In contrast to FoldX, Schrödinger software often presents energy values as negative numbers. For example:

ΔG(WT) = -18693.8 kcal/mol

ΔG(Mut) = -18712.4 kcal/mol

To calculate ΔΔG in this case:

ΔΔG=ΔG(Mut)−ΔG(WT)=(−18712.4)−(−18693.8)=−18.6 kcal/mol

This negative ΔΔG indicates that the mutant is more stable than the wild type.

3. Literature Confusion on the ΔΔG Calculation in the litterature:

ΔΔG = ΔG(WT) - ΔG(Mut) or ΔΔG = ΔG(Mut) - ΔG(WT)

is common in literature. The correct approach generally follows:

ΔΔG=ΔG(Mut)−ΔG(WT)ΔΔG=ΔG(Mut)−ΔG(WT)

This means that if you want to determine how much more stable or unstable a mutation makes a protein compared to its wild type, you subtract the wild type's energy from the mutant's energy.

So, when using FoldX, positive values indicate decreased stability, while negative values suggest increased stability. Understanding these conventions and calculations is essential for accurately interpreting protein stability data from different computational tools like FoldX and Schrödinger.

Best regards.

The confusion regarding the sign of the stability values when using FoldX or other computational biology software tools is understandable, as different programs may use different conventions for reporting energy values.

FoldX and Positive Values:

FoldX typically reports the stability of a protein in terms of the energy required to unfold it, which can be positive. This is because FoldX calculates the energetic changes in the protein structure, and a positive value indicates that energy is required to disrupt the protein’s structure. In FoldX, a higher positive value means the protein is less stable because more energy is required to unfold it.

ΔΔG Calculation:

When you’re comparing a wild-type protein to a mutant, the change in stability (ΔΔG) is calculated as the difference in the energy between the two states. The convention used in FoldX is:

ΔΔG = ΔG(Mut) - ΔG(WT)

This means if ΔG(WT) = 30 kcal/mol and ΔG(Mut) = 40 kcal/mol using FoldX, then ΔΔG = 40 - 30 = 10 kcal/mol. A positive ΔΔG value in FoldX indicates that the mutation has destabilized the protein compared to the wild type. Conversely, a negative ΔΔG value would indicate increased stability of the mutant compared to the wild type.

Schrödinger Software and Negative Values:

In contrast, Schrödinger and some other software tools use the convention where a more negative ΔG value indicates a more stable protein. In Schrödinger’s case, if ΔG(WT) = -18693.8 kcal/mol and ΔG(Mut) = -18712.4 kcal/mol, the ΔΔG would be calculated as:

ΔΔG = ΔG(WT) - ΔG(Mut) ΔΔG = -18693.8 - (-18712.4) = -18712.4 + 18693.8 = -18.6 kcal/mol

Here, a negative ΔΔG value indicates that the mutation has made the protein more stable, which aligns with the general thermodynamic principle that a more stable state has a lower free energy.

Conclusion on ΔΔG:

The correct formula for ΔΔG depends on the convention used by the software you are using:

For FoldX and similar tools that use positive values: ΔΔG = ΔG(Mut) - ΔG(WT)

For Schrödinger and similar tools that use negative values: ΔΔG = ΔG(WT) - ΔG(Mut)

When interpreting results, always refer to the software’s documentation or conventions to understand how stability is reported. Remember that the key is the change in stability (ΔΔG), not the absolute values of ΔG(WT) or ΔG(Mut).

Thank you so much for your kind response! Hicham Mechqoq Zhi Cao