Hi Jason,
Thanks again for your informed reply. I agree with the points you make re:
environment and coupling effects on the pKa's of the carboxylates in a
C-terminal glutamate / glutamic acid residue.
It was probably not appropriate to cite Thurkill et al. specifically here -
I just wanted to point out the likely order of pKa's of the side-chain and
C-terminal carboxylate in a C-terminal Glu residue.
Let me first say that I don't expect the residue to be important for
interactions or dynamics and that the NMR bundle points to a quite solvent
exposed (and 'disordered') C-terminal residue.
Then, from a quantum-mechanical perspective, the side-chain carboxylate of
a C-terminal glutamate would have a higher proton-affinity than the
C-terminal carboxylate, wouldn't it? The side-chain carboxylate is more
electronically 'isolated' than the C-terminal carboxylate, after all.
Wouldn't the coupling between the two carboxylate pKa's result in the
side-chain carboxylate shifting up and the C-terminal carboxylate shifting
down a little?
Predictions from propka3.0 on the models in my NMR bundle (which
incorporates coupling between different groups as well as the environment,
albeit in a relatively crude way of course) also point in this direction,
with the average pKa for the C-terminal carboxylate around 3.3 and the
side-chain carboxylate around 4.7.
I'd like to stay as close as possible (with standard MM MD) to the species
present in the NMR experiment; if I'd put a NME cap on the C-terminus, I
think I'd be further away from that than if I model the C-terminal
glutamate/glutamic acid as one of the possible (and probably most likely)
protonation states.
Thanks,
Marc
PS Francois, I saw your reply now too - thanks; I used 'extrapolate' for
want of a better word, and understand your dislike of using extrapolation
in relation to charge derivation! (hence the single quotes).
On 5 June 2012 18:29, Jason Swails <jason.swails.gmail.com> wrote:
> 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 Wed Jun 06 2012 - 00:30:02 PDT
