Gene Mutation and Analysis

Gene Mutation and Analysis

Gene Mutation and Analysis

Alignment and Mutant Identification

Gene Mutation and Analysis

Identifying the mutation is also automated using online bioinformatics tools that will align your wild-type amino acid sequence (the one you obtained from the PDB entry page in step 1) with your mutant amino acid sequence (your translation from the previous step). Note it is important to compare two protein sequences and not DNA to protein. For PDB entries with multiple chains, you should only be aligning the WT and mutant sequences of the indicated chain. The following link will perform a pairwise alignment of your protein sequences: https://web.expasy.org/sim/ Select User-entered sequence for each box. Paste your wild-type sequence in the top box and the mutant sequence in the bottom box. Make sure they are named accordingly. Use the default parameters and click submit. The two sequences will be aligned with wild-type on top and mutant on the bottom. If you have done this correctly, the first alignment will go the length of the entire sequence for both wild-type and mutant. It should have a score of 99.x% identity. If it says 100% you may have pasted the same sequence in both input windows or they may have been a problem with your mutant sequence and you should contact Dr. Roose. If you are getting many very short alignments with many gaps, you have aligned the wrong sequences (usually one DNA and one protein). Where they are identical will be marked with an asterisk and the location without an asterisk represents to mutated amino acid. If your eyes are having trouble finding the missing asterisk, use the find tool to search for ‘* *’.

 

Paste this alignment into an MS Word file with the mutation position highlighted. Adjust your margins if necessary to preserve the alignment formatting.

Mutant Identification

Identify the mutation by single letter code and position number using the following format H87A, where the first letter is the wild-type amino acid, the number is the amino acid position number and the second letter is the mutant amino acid. The numbers on your alignment represent position numbers within the sequence. You could manually count them out or use the find tool to identify the residue of interest in the wild-type sequence (+/- a few residues should be enough). Then highlight text within your FASTA sequence and go to Review –> Character count to determine the amino acid position.

Evolutionary Conservation Image and Sequence

Gene Mutation and Analysis

Go to https://consurfdb.tau.ac.il/index.php

 

Analyzing the evolutionary conservation of your protein structure will show what residues are variable and what residues the most conserved (or unchanging) across multiple sequences in different organisms. Residues that are considered variable can accommodate many different types of amino acids without changing structure and function. This means that these sites can mutate over time to different residues without any pressure to stay the same.  However, conserved residues means that the protein across multiple organisms tend to keep the same residue or residues with very similar properties. This is because there is selective pressure to keep residues with those properties or there will be detrimental consequences to the protein structure and function. This view can give you an idea of what regions of the protein are more important to structure and what regions are less important.

 

What are the 4 types of gene mutations?

Gene Mutation and Analysis

Click the link to the ConSurfDB to analyze your protein at the ConSurf Database. Input your PDB ID and select a chain from the pulldown list that autopopulates. (Just pick any one if your protein entry has multiple chains). Then click apply. Click (or download) the View Colored Sequence in PDF format. Screen shot your color-coded sequence and add this image to the slide. The results also provide a structure image with the same color-coding for the selected chain so you can visualize where areas of conservation/nonconservation are within the 3D context of your protein’s structure. Screenshot this image or select the ‘View ConSurf-DB results with full-size NGL viewer’ link at the bottom of your results to open a large view to save an image. Include this image on the slide.