Comparisons between amino acid sequences can be used to understand which areas of a polypeptide are important for its function. A large family of proteins in bacteria includes a transcriptional regulator called the cAMP regulatory or activator protein (CRP or CAP).
(i) Go to the NCBI site at www.ncbi.nlm.nih.gov/
(ii) Retrieve the amino acid sequences for the following CRP/CAP family proteins by selecting the Protein database in the Search menu and typing the underlined accession number into the adjacent box:
• E. coli catabolite gene activator (cAMP receptor protein), P0ACJ8
• Yersinia pestis cyclic AMP receptor protein, AAM87500
• Klebsiella pneumoniae cAMP receptor protein, A44903
• Shewanella sp. MR-4 Crp/Fnr family transcriptional regulator, YP_734144
• Aeromonas hydrophila fumarate and nitrate reduction regulatory protein YP_856826
(iii) For each retrieval, select "FASTA" from the display menu then copy and paste the amino acid sequence into a Word document, giving each sequence a recognizable byline in FASTA format: ">byline".
(iv) Remove the numbers from the amino acid sequence
(v) Go to the EMBL Clustal W site (www.ebi.ac.uk/Tools/clustalw/index.html), which will allow you to compare up to 10 different amino acid sequences.
(vi) Copy and paste the five sequences in FASTA format consecutively into the open box and hit RUN.
(vii) In a few moments, you will see an output that shows all five sequences lined up according to the best matches between them.
(a) Which parts of this protein show the most amino acid divergence?
(b) Which parts of this protein appear to be the most conserved?
(c) Go to www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi at NCBI. Paste in the E. coli sequence to see what domains appear. Do the same with the A. hydrophila sequence. What areas of the polypeptide are identified with the two smaller, common domains (colored in red and blue)? Comparing these two domains between these species, which domain appears more divergent? Does it make sense relative to the information given about each domain (scroll the arrow over each domain to retrieve information)? The e-value is a measure of similarity; similar sequences yield low evalues.