I'm interested in studying specific missense mutations in a human gene. My goal is to determine whether the mutated region of the protein is conserved across various species. Could you please guide me on how I can use in silico tools to find homologous protein sequences and identify their conserved regions?
Thank you very much
Obtain the protein sequence of interest from a reliable database such as UniProt (https://www.uniprot.org/) or NCBI Protein database (https://www.ncbi.nlm.nih.gov/protein/), Perform a BLAST search, Retrieve homologous protein sequences, Use multiple sequence alignment tools to align the protein sequences. Once you have the multiple sequence alignment, you can analyze the conservation of specific regions or residues. Conservation scores can be calculated based on various algorithms, such as Shannon entropy or sequence conservation scores. Tools like ConSurf (http://consurf.tau.ac.il/) and Jalview (https://www.jalview.org/) offer conservation analysis features.
That's a good approach Susanta Roy I would add that once you are working with your multiple sequence alignment (MSA) in Jalview (https://www.jalview.org), you load an experimental 3D protein structure, or an AlphaFold model (all possible from Jalview, just right-click on a sequence label), and visualise the mutations and conservation scores on the structure too. Jalview makes this easy by colouring the structure by the sequence, so you can choose to colour by conservation and add features to represent your mutations and they will instantly be viewable on the structure.
The other thing I would add is that in addition to BLASTing the full-length protein, have a look at it on InterPro and see what domains it has. Then you can work with curated MSAs from the individual domains too.
Great question Muhammad Abrar Yousaf !
Yes, there are several in silico tools and databases that can be used to find homologous protein sequences and identify their conserved regions. Here are the general steps you can follow:
Keep in mind that in silico tools have limitations and may not always accurately predict the effects of missense mutations on protein function. Therefore, it is important to combine in silico analysis with experimental validation to confirm thefunctional effects of missense mutations.
Dear Stuart Alexander MacGowan
Thank you very much for your guidance and appreciation. I am also getting the protein isoform sequences during BLAST. Can I also add them in multiple sequence alignment?
Dear Ahmad MohdAziz Hussein
Thank you very much for such detailed guidance.
Absolutely, Muhammad Abrar Yousaf ! It’s possible to include isoforms during alignment. However, one key aspect to consider is that isoforms may skew conservation scores, as the divergence between them represents a distinct process from that between homologs. A potential solution to this issue could be reducing sequence redundancy in the alignment. This can be beneficial in creating a representative set of sequences that are taxonomically distributed in such a way that conservation scores most accurately reflect evolutionary restraint. Fortunately, Jalview has a feature for removing redundancy, which simplifies this task!
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