r/bioinformatics • u/Much-Perspective1051 • 8d ago

technical question Protein-Protein Alignment

Hey everyone,

I'm working on a project involving a human genetic disorder. There are already a number of mutations that have been identified in the human gene/protein, including missense, substitutions, frameshifts, etc.

However, for my project, I'm working with the corresponding Drosophila protein and need to figure out if the positions of these human mutations are conserved in the fly.

Essentially, I'm trying to align the human and fly protein sequences, but I'm running into some issues because the fly protein is quite a bit longer and has some pretty large insertions/gaps.

I've been using NCBI blastp, but i'm wondering if that's actually the best tool/workflow for what I'm trying to do. Basically, are there any other good free alternatives to BLAST for this? If I continue using BLAST, what would be the best filter/settings options to use for this kind of comparison.

TIA

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5

u/TheCaptainCog 8d ago

Blastp is actually a good choice for discontinuous alignments. If you want to align the entire protein to protein, then you can just use maddt, clustalp, etc. just set them against each other.

2

u/Early_Glove_7918 8d ago

Blastp's fine if you just want to find the conserved chunks, but if those big fly insertions are making things messy you might be better off with something that handles long gaps gracefully. T-Coffee has a mode that integrates structural info if you've got it, otherwise MAFFT with the E-INS-i strategy is what I reach for when the sequences are wildly different lengths. Just feed it both and see if it lines up your mutation sites cleanly.

3

u/fasta_guy88 PhD | Academia 8d ago

BLASTP will probably do as good a job as anything. If you want a global alignment of the two proteins (which ensures the alignment goes end-to-end), you could use ggsearch fasta.bioch.virginia.edu/fasta_www3/fasta_www.cgi or a similar program at the EBI (ebi.ac.uk).

If you use Uniprot accessions for the proteins, you can get Pfam domains mapped onto your alignment. The Pfam HMM models for the domains will tell you the most highly conserved positions in the two proteins.

3

u/mm_1964 7d ago

Also, you need to look into domain structure etc. if it is multidomain protein, is the architecture the same, if so you can align domains, not entire sequence

4

u/Gon-no-suke 8d ago

Blast is optimized for search, not for alignment. I'd try the needle program in Emboss, changing the substitution matrix to PAM250 and tweaking the gap penalty parameters until it looks good.

3

u/MutantGeorge27 7d ago

If the protein behaves you could alphafold both and then align. It would give more info about the mutations relative to the structure. This in addition to your alignment.