Use BLAST to find structures in a mesophilic species that are sequence-similar to a query structure in a thermophilic species
Analyze the sequence conservation of a binding site between the two structures
Use VAST to build a structural alignment between these structures
Analyze the VAST alignment to discover structural differences that may confer thermostability
Steps
Find sequence-similar structures with BLAST
From the NCBI home page, click the BLAST link on the top tool bar.
Click protein blast.
Set up the BLAST search
Enter 1IQR_A in the Enter Query Sequence box (a T. thermophilus DNA photolyase).
Under Choose Search Set, select Protein Data Bank proteins(pdb) from the database menu.
In the organism box, type select Escherichia coli (choose taxid:562 from the list).
Click the BLAST button to begin your search.
View the BLAST results
When your search is done, scroll down beneath the graphic summary, and make a note of the PDB code of
the best E. coli homolog.
Use VAST to find structure neighbors to 1IQR
Return to the NCBI home page, and click Structure on the top tool
bar.
Enter 1IQR in the search box and click Go.
Click on the accession to load the structure summary page.
Click on the gray bar labeled Chain A to load the VAST neighbors.
View the VAST alignment in Cn3D
Type the PDB code (1DNP) for the E. coli sturcture in the Find box and click Find.
Check the box to the left of chain A of the E. coli structure (make sure you choose the entire chain, the row with the longest alignment).
Click "View 3D Alignment".
Analyze the co-factor binding site
Select Style / Coloring Shortcuts / Object. Highlight the FAD in 1IQR and find the
residues within 3.0 Angstroms of the ligand.
Do the same residues contact FAD in both structures?
Are the residues that do contact FAD in both structures conserved?
Locate potential features conferring thermostability
Choose Style / Coloring Shortcuts / Secondary Structure (helices are
green, strands are tan, and loop/coils are blue).
Which sequence has more gaps (represented as ~ symbols)?
In what kind of secondary structure element do they occur most frequently?
Locate potential features conferring thermostability
In the sequence window, choose View / Find pattern, and search for PPP (proline triplet).
Locate any matches. In which sequence is it? Are there other prolines nearby?
View these residues in the structure. Proline-rich sequences tend to form polyproline helices. Do you see any evidence of that happening here?
Develop a hypothesis
Using the evidence gathered in the last step, form a hypothesis about how the T. thermophilus structure may be more stable at high temperature than the
E. coli protein.
