MFE tutorial: Extra Tool to help analyze pK.out
In this tutorial, we use the results of a pKa calculation to perform a more detailed analysis of a specific residue, AspA170, in Photosystem II from pea plants (PDB ID: 5XNL). The goal is to move beyond raw pKa values and examine the energetic factors that determine the protonation state of this residue.
It is absolutley neccesary that you run a pKa calcualtion to obtain the neccesary files to be able to use this tool.
Background
Photosystem II is a large protein supercomplex that initiates the photosynthetic electron transport chain in oxygenic photosynthetic organisms, including cyanobacteria and higher plants such as pea and spinach. At the core of this complex lies the oxygen-evolving complex (OEC), a metal cluster responsible for catalyzing the oxidation of water to molecular oxygen.
The OEC carries a high positive charge and therefore requires stabilization through coordination by negatively charged amino acid residues. In this tutorial, we use the MFE program to evaluate whether one such ligating residue, AspA170, energetically favors the negatively ionized state, thereby assessing its contribution to the stability of the metal center.
What does MFE do?
MFE (mean field energy) calculates the mean field ionization energy on an ionizable residue at a specific pH/eH. MFE provides the energy interctions of ionized residues and its neighbors.
Files needed
Files needed to run this tool is fort.38, head3.lst, pK.out, and sum_crg.out.
Usage
If you have succesfully installed the MCCE-Tools you should be able to call the tool from any directory.
mfe.py -p 7 -c 0.05 ASP-A0170_
The -p flags defines at what pH the analysis is done. -c is at the energy cutoff that defines the energy minimum for residues interaction.
Output
Output for the analysis should look like this for ASPA170 on 5xnl (Pea Photosystem II in yrh D1 subunit near the OEC):
Residue ASP-A0170_ pKa/Em=2.358
=================================
Terms pH meV Kcal
---------------------------------
vdw0 -0.00 -0.09 -0.00
vdw1 -0.30 -17.25 -0.41
tors -0.25 -14.38 -0.34
ebkb 1.72 99.83 2.35
dsol 10.85 629.88 14.80
offset -0.62 -36.17 -0.85
pH&pK0 -2.25 -130.60 -3.07
Eh&Em0 0.00 0.00 0.00
-TS 0.00 0.00 0.00
residues -13.13 -761.88 -17.90
*********************************
TOTAL -3.97 -230.66 -5.42 sum_crg
*********************************
CTRA0040_ 0.10 6.08 0.14 -1.00
NTRA0043_ -0.07 -3.93 -0.09 0.99
ASPA0059_ 2.39 138.57 3.26 -1.00
ASPA0061_ 5.94 344.57 8.10 -1.00
ARGA0064_ -1.42 -82.26 -1.93 1.00
GLUA0065_ 2.02 117.29 2.76 -1.00
ASNA0076_ 0.07 3.86 0.09 0.00
THRA0085_ 0.15 8.84 0.21 0.00
SERA0086_ -0.06 -3.72 -0.09 0.00
HISA0092_ -1.36 -79.08 -1.86 1.00
Conversion of units
This output gives you the a varierity of the interactions of energy terms in differen units (pH, meV, kCal).
At 298 K (room temp):
Energy in meV (single proton): • ΔE(meV) ≈ 59.16 * ΔpH
Energy in kcal/mol: • ΔG(kcal/mol) ≈ 1.364 * ΔpH
Definitions of terms
1) Vdw0: vdW interactions within the conformer (a side chain or ligand) + interaction with implicit solvent
2) Vdw1: vdw interaction of this conformer with the protein backbone
3) tors: torsion energy of the conformer
4) dsol: Loss of solvation energy compared with this conformer in solution. It should be positive as it is a loss in energy.
5) offset: energy term that can be freely modified
6) pH&pK0: Solution pH effect on ionization. It is the environmental pressure on residue ionization. For an acid, low solution pH makes ionization (releasing a proton) easy, so it contributes favorable energy. For a base, low pH makes ionization harder. When pH equals the residue’s solution pKa, the environment pH is at a balance point, where the contribution is 0.
7) Eh&Em0: Environment Eh effect on redox reaction. This works similarly to pHpK0.
8) -TS: Entropy term.The number of rotamers of neutral and ionized residues generated by MCCE may differ. The effect of different rotamer counts on the two ionization states acts like entropy.
9) residues: Total pairwise interaction from other residues. Other residues may shift the ionization free energy depending on their dipole orientation and charge.
10) total:Total pairwise interaction from other residues.
Interpratation of the Data
The purpose of the MFE calculation is to assess whether the ionized or neutral state of ASPA170 is energetically favored at a given pH. Positive MFE values indicate stabilization of the neutral form relative to the ionized form, while negative values indicate preferential stabilization of the ionized state.
In the results shown above, the van der Waals contribution slightly favors the ionized state of AspA170 in Photosystem II. More importantly, the total pairwise interaction term strongly stabilizes the ionized form. This outcome is chemically intuitive, as AspA170 must remain negatively charged to effectively coordinate the highly positively charged ions of the oxygen-evolving complex (OEC).
The lower portion of the table reports individual pairwise interactions between AspA170 and neighboring residues. These contributions can be examined in the same manner to identify which local interactions most strongly influence the residue’s protonation state.
We begin by examining the terms with the largest absolute values in the energy column (e.g., kcal/mol). These dominant contributions typically arise from two sources: 1. Residue–residue interactions 2. Desolvation energy (dsol)
In this example, favorable interactions with neighboring residues strongly oppose the unfavorable desolvation penalty. Importantly, these stabilizing interactions do not merely offset the penalty; they exceed it, yielding an additional ~6.6 kcal/mol of net stabilization.
This interplay between opposing energetic terms is the central purpose of an MFE (mean force energy) analysis. Rather than focusing on individual large energy values in isolation, MFE analysis aims to explain why a residue adopts a particular charge state or pKₐ under the conditions studied (even when the pKₐ itself is not explicitly reported). By decomposing the energetic contributions, MFE reveals how the local environment stabilizes or destabilizes specific ionization states and thereby shifts protonation equilibria.
In practice, large desolvation penalties are common for buried or functionally important residues. What allows these residues to remain charged is the presence of compensating local interactions—precisely the balance that MFE analysis is designed to quantify and interpret. From the total interaction energy, we can reasonably conclude that the ionized conformer of AspA170 is favored over the neutral form, as indicated by the negative value. This preference is beneficial for the system, since maintaining a negatively charged AspA170 helps stabilize the highly positively charged oxygen-evolving complex (OEC).