Re: [AMBER] protonation of protein-ligand complex

From: Jason Swails <>
Date: Thu, 14 Nov 2013 16:29:27 -0500

On Thu, Nov 14, 2013 at 2:30 PM, Thomas Evangelidis <>wrote:

> Dear AMBER community,
> I am simulating a protein-complex in which protonation of two glutamic
> acids in the binding pocket is believed to dictate ligand binding and
> release. I want to calculate pKa values from conformations along the
> simulation and change if necessary the protonation of the protein by also
> taking into account the ligand. I used for this purpose PROPKA 3.1 and H++
> servers, but the results are contradictory. According to PROPKA 3.1 the pKa
> values of the two Glu residues in the starting conformation are 8.07 and
> 7.61, therefore in pH 7.6 both will be protonated.

I disagree here. The first Glu will be protonated in about 75% of the
ensemble and the second Glu will be protonated in ~50% of the ensemble.
 The pKas are far too close to the pH for a single protonation state to be
considered 'dominant'.

According to the
> titration curves produced by H++ these two residues are never protonated at
> pH 7.6 whatever dielectric I use for the protein (tried values 1-10). It is
> worth noting that the titration curve of the first Glu deviates a lot from
> the Henderson-Hasselbalch curve.

This could mean that either the residues do not follow HH titration (if
they are coupled to ionizations of other residues, for instance), or it
could mean that the model is incapable of correctly predicting the
ionization equilibria (or both).

> I am confused about what results I should trust.

Neither. A pKa of 7.5 to 8 for a glutamate is very high (it's about a 3 to
3.5 pK unit shift from the pKa of an isolated Glu in solution). There are
no methods currently out there that can reliably predict a pKa shift that
large. And pKa shifts in membranes have only recently been looked into.

I'll answer more of your questions in other emails.

Jason M. Swails
Rutgers University
Postdoctoral Researcher
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Received on Thu Nov 14 2013 - 14:00:02 PST
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