Background: Proteins bind ligands/substrates through molecular interactions provided by specific amino acids in the binding pocket. These...

Background:

Proteins bind ligands/substrates through molecular interactions provided by specific amino

acids in the binding pocket. These interactions (e.g. hydrogen bond, hydrophobic interactions,

and electrostatic interactions etc.) are key to binding and in turn modulating the proteins

function. As a result, one common drug design strategy is to design molecules that would

make favorable interactions to these essential amino acids in order to outcompete the native

substrate and inhibit the protein's function. At the same time, regions that can change can be

important to take advantage of for designing organism specific drugs and avoiding off-target

effects (e.g. maybe you want the drug inert in humans but highly functional in dogs). In this

case you want to take advantage of the sequence differences between species. In this lab, you

are going to identify and visualize these important amino acids through protein sequence

analysis using bioinformatics tools.

Instructions:

1. We are going to use a sequence visualization program (Jalview). This is where we can identify

the residues we think are interacting with the molecule in the active site.

2. On the page of your protein from the pdb website, click on fasta sequence under the "Display

Files" menu.

3. This will display the fasta sequence on your browser (which is the amino acid sequence for your

enzyme).

4. Copy this sequence then go to the BLAST web server:

5. Select Protein Blast (blastp)

6. Paste in your sequence. Wait! Don't hit submit, instead lets add some paramters to make sure

you have enough diversity in sequences to get a good analysis of conservation:

a. Change database to: Reference Proteins (refseq_protein). This database is a bit less

redundant than the default nr database. Ideally we would use a database that is nonredundant

on the 75% level (other servers such as HMMER allow that, but they are more

difficult to use).

b. Expand "Algorithm Parameters" and select 250 on "Max Target Sequences". This will

make sure you have enough sequences to measure conservation while making sure that all the

proteins identified are predicted to be highly related.

*IMPORTANT NOTE: If you see %'s in the "Ident" column in the blast results

going below 30% re-adjust this to 100 or even 50. Below 30% you are in what is

considered the "twighlight" zone and the sequences you are finding are not

necessarily related in structure or function. Basically, you have drifted too far in

evolutionary space. Roughly speaking, 30-70% Ident it has been found that proteins

are both related in protein structure and function, above 70% it is almost certain that

proteins are almost identical in structure and function.

7. Hit "BLAST" to initiate search results

8. Analyze your results, make sure all the hits are "Pink or Red" for alignment score (i.e. large

portions of the sequence aligned). And in the "Descriptions" table with "Sequences producing

significant alignments" make sure the Ident column never goes below 30. If it does, see note above.

9. If all looks good, Select "Multiple Alignment" near the top in the "Other Reports" section.

10. From this output (don't worry if an error is thrown for graphical overview) select:

"Download" -> Fasta plus gaps

13. Now we are going to open another program named Jalview (. Click

Launch Jalview Desktop open the downloaded file.

14. Under the "File" menu, hit Input Alignment, from file and open the alignment file you just

downloaded from the BLAST.

15. Under Jalview hit colour tab at top, then click percentage identity, now go back to

color and click above identity threshold. Change occurrence to 99% conserved.

15. The residues highlighted here are either critical for protein structure or function. All of the

residues you identified in lab 1 (i.e. the catalytic residues) should be highlighted.

16. Using Jalview and using percentage identity and moving the bar back and forth you can

determine the percent conservation for a residue (look at switch from being

colored to not colored).

17. Now, in the assignment below you will need to find non-catalytic residues (i.e. residues not

directly involved in the reaction mechanism) that are either highly conserved OR have low

conservation. Roughly speaking (based on search paramters above):

High Conservation residues have >70% identity across the homologous sequences

Low Conservation residues have <30% identity across the homologous sequences

Residues in the 30-70% range a likely important but not critical for structure or function.

Assignment---

For your assigned enzyme group complete the following assignments with the help of the Lab

2 instructions:

1) What NON-CATALYTIC residues in the active site of the enzyme are most likely

important for function?

a. Highlight three highly conservative residues, show both structural interactions

AND conservation using PyMol and JalView. Hypothesize why they are critical for

function.

b. Highlight one low conservation residue, show both structural interactions AND

conservation using Pymol and JalView. What other amino acids are observed,

hypothesize why the alternate residues are ok.

2) How does the inhibitor interact and how would you further optimize?

a. Describe WHY it acts as an inhibitor.

b. What key interactions are made with the inhibitor? Are any of those interactions

mimic the natural substrate?

c. If you were to modify the inhibitor what new interactions would you try to take

advantage of? (hint...conserved residues in the protein are unlikely to change

while maintaining protein structure or function)

Case 1 = ACE: 2X91

Case 2 = OD: 2TOD

Case 3 = α-Fucosidase: 2ZX5

Case 4 = PNP: 1A9S

Case 5 =CD: 2FR6