On Tue, Jun 5, 2012 at 11:46 AM, Marc van der Kamp <marcvanderkamp.gmail.com
> wrote:
> Dear Jason and Francois,
>
> Many thanks for your replies.
> Francois, I was thinking of taking charges from GLH in amino12.in, and
> adjusting the charges in line with the changes between GLU in amino12.inand
> aminoct12.in. In this way, the issues you mention regarding differences
> between carboxylic acid and carboxylate don't apply for the side-chain. I
> already noticed that changes in GLU charges in amino12.in and
> aminoct12.inare quite small, and therefore thought that
> 'extrapolating' these changes
> for a C-terminal GLH might be OK.
>
> Jason, I want to run standard MD at a pH around 4 (not uncommon in NMR
> experiments, for example). Under those conditions, one would expect a
> (solvent exposed) Glu side-chain to be (partly) protonated (pKa ~4.25)
> whereas a C-terminal carboxylate may well be unprotonated (pKa ~3.67, see
> e.g. Thurkill et al., doi: 10.1110/ps.051840806).
> So, I didn't think that a C-terminal GLH is so strange, at least not within
> the limitations of standard MM MD.
> But perhaps there is a good reason that C-terminal GLH is never present in
> standard AMBER forcefields?
>
I'll agree that this is a tricky situation, but I think the assumptions
you're making above are dangerous. The pKas that are quoted are for
solvent-exposed residues that are not shifted significantly compared to
free amino acids (since environment can have a strong perturbing effect on
pKas). Also, all of the amino acids they titrate (at least in the
Thurlkill paper) are capped amino acids, because they did not want the
termini affecting the pKas (and they would).
The two carboxylates on a C-terminal GLU are coupled, and their pKas cannot
be estimated independently, IMO. Therefore, I would argue that the pKas
you cited for C-termini and for GLU do not apply specifically to a
C-terminal GLU.
Ultimately, if the C-terminal residues is very important for dynamics or
some crucial interaction, you will probably need to determine a better way
of treating it (alternatively, can you just cap the C-terminus with a NME
group?). Otherwise, I would suggest just using CGLU. You may be able to
use your proposed CGLH also to show that the protonation state is not vital
to the behavior of your protein (or maybe a double-protonated C-terminal
GLU).
Of course both Francois and I have our biases in this case -- he's an
expert on charge derivation and I study ionization equilibria and pKa
calculations in proteins.
HTH,
Jason
--
Jason M. Swails
Quantum Theory Project,
University of Florida
Ph.D. Candidate
352-392-4032
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Received on Tue Jun 05 2012 - 11:00:03 PDT
