Subject: Re: CCL:MEAD pKa calculations

From: Don Bashford <[email-masked]>
Date: Thu, 13 Feb 2003 12:32:32 -0800

>>>>> "CUI" == CUI, Guanglei <[email-masked]> writes:

    CUI> Dear CCL readers, I'm trying to use MEAD to calculate pKa of
    CUI> three residues in a ligand binding site. The structure has
    CUI> been minimized with AMBER force field with explicit
    CUI> solvent. The charges of these residues have been derived from
    CUI> RESP calculations. All needed files are created and multiflex
    CUI> calculation used epsin = 4.0. After the calculation, the
    CUI> intrinsic pKas of two residues are very surprising. So here's
    CUI> my questions:

Hopefully you've used a fine grid of 0.25 Ang or so centered on
"center of interest".

    CUI>      1. What could be reasons that the self interactions for
    CUI> residue UNK and LYS are very big.  

Probably because the accessibility of these residues to solvent is
low.  This tends to disfavor the charged states, and therefore lowers
pKint for Lys, and raises it for things like Asp and Glu.

    CUI> 2. During the multiflex
    CUI> calculation, I was warned of "vertex found with count = 2"
    CUI> three times. How should I deal with this?  

This is almost certainly harmless.

    CUI> 3. Do I need to
    CUI> include explicit solvent molecules in the calculation,
    CUI> generally speaking? The binding site is not completely
    CUI> buried.  

People have done this both ways.  It can be argued that keeping
crystallographic waters is more "detailed", but on the other hand,
when the ionization state of a residue changes these waters may move
or reorient, and a calculation that regards them as having fixed
coordinates will not capture this.  A blob of high dielectric can at
least repolarize!  In my experience, ordinary protein residue pKa's
are not much improved by including crystallographic waters, and may
even get a bit worse.  However, for multivalent things like metal
centers, an explicit hydration shell helps, because the hydration
shell is tightly bound and a singe change change doesn't really
re-orient it so much.

    CUI> 4. LYS (protonated) has 3 equivalent hydrogens. It's
    CUI> not very clear to me which one should be zero charged in
    CUI> LYS.st file.

My guess is it won't make so much difference, but why not try it all
three ways?
     
    CUI>      Here're the .summ file. Sorry for the lengthy
    CUI> description. Any suggestions are welcome. Thanks in advance.

    CUI>  site name           pKmod      delta self    delta back      pkint  
    CUI>    UNK-259           7.8       23.1134      -5.41211       25.5013 
    CUI>    LYS-162          10.4      -11.4993       1.32968        0.23042
    CUI>    TYR-155           9.6      0.694971      -1.28149        9.01348

What is UNK?  I'll assume it's negatively charged.  It's delta self is
indeed quite big.  It may be deeply buried.  It's charge may be very
compact or large (-2?).  The delta self you get is rather typical of a
buried lysine.  Do UNK and Lys interact strongly?  (see the .g file)
If so, the favorable charge-charge interaction may stabilize the
charge pair in spite of the large delta self terms.  I've seen such
things happen, for example in bR (Spassov et al., 2001, JMB 312:203).

Cheers,
Don Bashford

---

From: "CUI, Guanglei" <[email-masked]>
Date: Thu, 13 Feb 2003 23:29:23 -0500 (EST)

Thanks for the reply. 

> Hopefully you've used a fine grid of 0.25 Ang or so centered on
Yes, I did so, focusing to residue UNK, which is of my interest. It's
neutral and has one phenol group. 
> pKint for Lys, and raises it for things like Asp and Glu.
All three residues are inside of the protein with no solvent
accessibility. 
> My guess is it won't make so much difference, but why not try it all
One of the three is different from the other two. pkint is calculated
around 8. Very interesting ... 
> things happen, for example in bR (Spassov et al., 2001, JMB 312:203).

UNK is triclosan. It's neutral. The phenol hydroxyl hydrogen is
hydrogen-bonded to the TYR in hydrogen-added crystal structure. The LYS
is the only titratable group sticking inside of the protein. From the
great impact of hydrogen atoms of the LYS, I guess the same effect could
happen to UNK too. Here's the .g file. All terms are fairly small. 

1 1   0.000000e+00
1 2   2.158370e-02
1 3   4.047996e-02
2 1   2.158370e-02
2 2   0.000000e+00
2 3   2.034296e-02
3 1   4.047996e-02
3 2   2.034296e-02
3 3   0.000000e+00

-- 
Guanglei Cui
Dept. of Chemistry
SUNY at Stony Brook
Stony Brook, NY 11790