[gmx-users] Mutagenesis

Maik Goette mgoette at mpi-bpc.mpg.de
Wed Aug 8 11:25:12 CEST 2007


The question arises, is "physically acceptance" equal to "biological 
reality" and the answer is (with reasonable probability) no.

What I suppose, you want to do is calculating the affinity difference 
(free energy difference) for binding of a slab of DNA to a protein with 
"mutated" aminoacids.
Therefore you will calculate via TI or so the difference in free energy 
of two states, where A is the one with the original AA and B with the 
"mutated" AA. With GROMACS, you can morph between these states and 
calculate the DeltaG.
To get the atoms of the B-state positioned correctly, you may setup a 
library of your AAs and fit the backbone atoms to the BB-atoms of the 
original one. Then you have more or less reasonable positions for you 
B-state side-chain atoms. Afterwards have a look which side chain atoms 
of the B-state AA you really have to grow and afterwards start the sim.
Which kind of TI one should use is hard to say, but I'd suggest discrete 
TI (not slow growth), because your B-state has the time to equilibrate 
(more or less) properly.....

If all this is feasible, one probably can't answer. At last, that's the 
best one can do....and, btw., expect to invest some time to get this 

Hope, this helps.


Maik Goette, Dipl. Biol.
Max Planck Institute for Biophysical Chemistry
Theoretical & computational biophysics department
Am Fassberg 11
37077 Goettingen
Tel.  : ++49 551 201 2310
Fax   : ++49 551 201 2302
Email : mgoette[at]mpi-bpc.mpg.de
WWW   : http://www.mpibpc.gwdg.de/groups/grubmueller/

Esther Caballero-Manrique wrote:
> Hi everyone,
> I am new to mutagenesis studies, so I was wondering if I could get some 
> input on my approach. My task involves mutagenizing a residue (with all  
> 19 possibilities)  in a protein that binds DNA, and then deciding 
> whether the resulting structure is physically acceptable.
> My approach to do this is 1) mutate using MODELLER,
> 2) check the structure with something like PROCKECK (although since I am 
> just mutating one residue, this doesn't seem important/useful),
> 3) align the resulting structure from MODELLER with the original and 
> paste the DNA
> 4) check for clashes with PROCHECK, and
> 5) do MD to see whether the model is feasible/calculate free energy of 
> binding.
> Obviously all steps are easy and fast except the last one, and my 
> question is, does anyone think that step 5 is overdoing it if one just 
> needs to know whether a structure is feasible ( i.e., I am not using MD 
> for refinement, but as a check of the feasibility of the complex)? Does 
> anyone have a better/easier way to do this?
> Thanks a lot for your help,
> Esther
> -- 
> Esther Caballero-Manrique
> Unit of Cancer Pathology
> Center for Excellence in Research on Aging
> University "G. D' Annunzio"
> Via Colle dell' Ara
> 66013 Chieti Scalo (Chieti), Italy
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