To answer all your questions, a simple number might not be enough. The structure of your protein has been determined experimentally (PDB id 3D30). So the best thing to do is to look at the structure (using pymol, for example) and see what effect your substitutions might have. You introduce the mutations directly using Pymol and see if there are any major clashes. You can also produce your mutant using SwissModel. Then superimpose the mutated and wt structure to see if there are any major distortions. And check in literature if known binding sites are affected. I quickly eyeballed your mutations: I'd say A/M=ok; P/G=loop distorted; T/A=no structural effect; P/T=loop slightly distorted; W/A=structurally probably ok, however this surface WW motif has certainly a function. In the crystal structure the W mediates tetramerisation, and this would be affected by the mutation; V/E: can be accommodated structurally, but is close to WW motif, and might be implicated in interactions; V/F=inside protein core, the larger F will lead to distortions and destabilise protein. Let me know if you need any further explanations/suggestions.

Hi Mahendra,

You can investigate the effect of point mutation using the FoldX tool.

As mentioned in my previous answer to a related post (https://www.researchgate.net/post/Can_anyone_suggest_why_the_wild_type_protein_structure_is_unstable_compared_to_its_point_mutants_in_my_protein-protein_docking_study), you can compute the structural perturbation of each point mutant as follows:

ΔΔG(change) = ΔG(MT) - ΔG(WT)

where ΔΔG(change) > 0 : the mutation is destabilizing and ΔΔG(change) < 0 : the mutation is stabilizing. (Equation taken from http://foldxyasara.switchlab.org/index.php/Training_exercises).

For details on installation and tutorial, go to:http://foldxyasara.switchlab.org/index.php/Main_Page

http://foldxyasara.switchlab.org/index.php/Main_Page 

Thanks Chanin,

I will try FoldX

To answer all your questions, a simple number might not be enough. The structure of your protein has been determined experimentally (PDB id 3D30). So the best thing to do is to look at the structure (using pymol, for example) and see what effect your substitutions might have. You introduce the mutations directly using Pymol and see if there are any major clashes. You can also produce your mutant using SwissModel. Then superimpose the mutated and wt structure to see if there are any major distortions. And check in literature if known binding sites are affected. I quickly eyeballed your mutations: I'd say A/M=ok; P/G=loop distorted; T/A=no structural effect; P/T=loop slightly distorted; W/A=structurally probably ok, however this surface WW motif has certainly a function. In the crystal structure the W mediates tetramerisation, and this would be affected by the mutation; V/E: can be accommodated structurally, but is close to WW motif, and might be implicated in interactions; V/F=inside protein core, the larger F will lead to distortions and destabilise protein. Let me know if you need any further explanations/suggestions.

Dear Mahendra,

as per your question r u interested to uncover the effect of point mutations on structural level/functional level of the protein. Is't point mutations responsible for gain of function/Loss of function.

for that u can perform a simple task

1) To predict basic parameters of both wild type and mutant  protein by using Protparam tool.

web.expasy.org/protparam/

compare basic parameters

2) secondary structure prediction of both wild type and mutant by using SOPMA tool.

compare result as per number of secondary structures presents and its positions along with sequence (Motif, Domain and Fold).

3) 3D structure prediction of both proteins ie Wild type and mutated protein by using molecular modeling tools or servers (swissmodel,phyre,rosetta).

4) u can directly compare the 3D structure of wild and mutant protein by using Dali lite server.

http://www.bioinfo.biocenter.helsinki.fi/dali_lite/start

then infer function from structure by using suitable data mining tools. and conclude whether point mutations are responsible for gain of function/loss of function.

Very very thanks to Mr. Stefan Arold  and Mahadev Asaram Jadhav

If i simulate both proteins (wild and mutant) for 0.5/1 ns using GROMACS and then analyse them by using energy, RMSD etc parameters...

Am i enable to find out difference in their 3D structure?

Dear Mr. Stefan

How do you identified this result could you explain and by which command you use in pymol.

Yes, MD simulations might be a good approach. I'm not sure that 1 ns is enough, though. And make sure that there is no biologically significant multimer that you miss -- see literature. I would just use swiss model to produce the mutant protein, and then see how this mutant compares to wild-type. It's much faster, and probably just as informative if you have the 'structural eye'. 

Desmond.  Mutate the aminoacids and relax the structure with the relaxation protocol.  It will give you 3D info.

I just had another quick look. In case you really wish to determine some sort of overall rmsd number, then yes you can run MD simulations (I suggest to use CHARMM force field, not GROMACS). Depending on what resources you have, this may take days or weeks. This number will probably be pretty depending on programme and parameters used. However if you want to understand the functional and biological effect of your substitutions, then you simply do swiss model (see attached file), compare with wt crystal structure (use 4FER), read the literature (georgelis et al PNAS 2012) and analyse. Takes less than 1h. You can quickly see that the W/A substitution will hugely affect the cellulose binding of your protein (which you won't see in MD). The V/E substitution is close and might also negatively affect cellulose binding. The other substitution appears actually ok, but you need to check in literature about functional surfaces in your protein. For example T/P and T/A are in a cleft in the protein that might have a particular function. As for Pymol commands: 'align' in the Action tab, and for the mutations: Wizard>Mutations; select target mutation in right hand of display, and cliock on residue to be mutated. Then use left and right arrows of your keyboard to change rotamers. You'll see the red discs appearing in places of clashed.

You first need to figure out which specific questions you wish to address. How ligand binding is affected by the mutation? Which ligand exactly? Will the mutant structure be as stable as the wild-type structure? The free energy perturbation (FEP) method may help to address these questions? ~150 ns simulation times should be a good compromise between rigour and resources. A question like What would be the structure of the mutant would require different approaches and much longer simulation times.