Predicting the effect of mutation on a membrane protein, structure and function.

Question

[Samane]

Edit (to be a fit for the site):

When cloning an integral membrane protein (~150AA/~462bp), two AA changes were introduced at the N or C terminal of the sequence, assuming these changes are real and not due to sequencing errors, which computational methods exist to predict:

• the effect of the mutations on membrane integration
• the 3D-structure
• the function of the protein

(now sounds like a homework question;)

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Hello everybody, I cloned my gene with gibson asembly method, but after cloning and sequencing I got two point mutation in my sequence ..that bot of them are at the end of my protein sequence, which Isoleucine exchanged to Methionine. I want to check this can be effect on my protein structure and function or not? how should I check it ?

I am looking forward to your kink response.

Sincerely yours,

Samaneh

[Joe]

Is there already a resolved structure for your protein?

Are you certain your SNPs are real? If your sequencing was Sanger based (which it often is for long sequences/cloning confirmation) it can be prone to errors, especially near the ends of the sequence, make sure you check the chromatogram to be sure those are reliable SNPs. Do you see them in any of your other clones?

[Samane]

thank you,

actually my protein is not too long and it is about 462 bp. and I cloned into the vector to expressed it on the cell surface. I checked the chromatogram by Chromas software, it was true and there is not any noise in that position. I don't want clone it again, therefor first I want to check it is important mutate or not?! actually it is weird to me, because I worked with Phusion enzyme that it has a proofreading feature ;/ and I don't know why it happened, also I send more colonies for sequencing and most of them was the same

[Michael]

Hello Samane!

We believe that this post does not fit the main topic of this site.

This looks like a biology question and has no apparent connection to something bioinformatics could help you with.

For this reason we have closed your question. This allows us to keep the site focused on the topics that the community can help with.

If you disagree please tell us why in a reply below, we'll be happy to talk about it.

Cheers!

Answer 1

[Joe]

This question doesn't really belong here, hence why it's been closed - however, if your protein is a membrane protein I think you can pretty much forget simulating its structure. You might be able to get a reasonable structure with Phyre2 or LOMETS by threading your sequence on to a published structure, but that will only work if there is a good published structure in the databases. Beyond that we really can't help you any further - so good luck!

[Michael]

I think you have found the bioinformatics aspect to it, but the question will have to be re-formulated to fit. Note that OP didn't answer the question about the 3D structure.

Answer 2

[Michael]

In this case it is really, really important to not withhold relevant information about your work. Why are you reluctant to share this information?

Given your hints 462 bp (or AA?? 152aa is really small) and membrane associated, I assume it is some kind of receptor. It possibly has a transmembrane helix for membrane integration, you could use a tool like TOPCONS and see if it changes between the two sequences. If not, you have at least a hint that it might not affect the membrane integration. Possibly, the mutation is intracellular and the rest is extracellular, then you could be safe.

Further

• Do you know the functional site of the protein? If it yes, the mutation should be far away from it.
• Compare the chemical properties of the AA's, in this case both are non-polar.

[Samane]

thank you very much for your help. my question was not biologic I don't know why it closed, because I wanted to know about a bioinformatic tools that compare two protein structure to each other not any more. coz always I did by my own and it take time and after I find a bioinformatic online tools that was so simple
I gave more biologic detailed to jrj.healey's response.

thank you so much for your advise. I will search again.

[Samane]

my protein name is VVD ( a photo switchable protein) that I clone in pDispaly vector which has a integrin protein membrane in its backbone then I just cloned my fragment into this protein. it is the biological part.( just foe explanation) now I want to see and check the functionality of my protein that if this point mutation will affect in my protein functionality or not? sincerely yours,

and again thank you for your quick response.

[Michael]

Great, so this is essentially a fusion protein. For both, parts - integrin - and VVD there seem to be 3D structures in PDB. Now you can submit the original and the mutated AA sequences of the protein to i-Tasser, and compare the models that come out, look for large differences. I think that is the best we can do at the moment.

[Joe]

Another option might be a dedicated homology modelling server such as MODELLER. You'd need to create alignments of your sequences first, but that's easy enough with Clustalo and other tools. ITASSER kind of does this anyway from what I understand so it's probably just as easy to use that.

[Samane]

thank you so much for your help.