Biochemistry 601

September 1, 1999 Analysis of Hydropathy and Sequence Conservation

You will need to use the ClustalW sequence-alignment software either by remote execution or on your own CPU.  You can obtain the ClustalW software in a zip file that must be saved to disk, unzipped with pkunzip, and then the resulting self-extracting file executed.

Assignment

1. Characterize the most hydrophilic and most hydrophobic 9-residue segments of a protein
• Is the segment buried or exposed?
• Is it in an alpha helix or a beta sheet or other secondary structure?
2. Characterize the most conserved 9-residue segment of a protein.
• Is it buried or exposed?
• Is it near an active site?
3. Make a brief presentation to the class about a protein (not necessarily the same protein used for parts 1 and 2), providing the following information:
• Name, function
• Taxonomic distribution
• Subcellular localization
• Principal structural features, including secondary structure (alpha, beta, alpha/beta, alpha+beta, irregular) and fold (TIM barrel, etc.)
• Quaternary structure
• Any known or speculated structure-function relationships (e.g., rope-like property of collagen triple-helix)

• Identify a protein having the following two (2) characteristics
1. the crystal structure is available in the PDB
2. homologous proteins have been characterized for organisms in two kingdoms which are different from the one containing the organism that yielded the PDB file.
• For example, my PDB file is for the Saccharomyces cerevisiae (yeast) enolase. It's PDB ID code is 1EBG. Luckily, the PDB file contains its Swiss-Prot entry name: ENO1_YEAST. I check the Swiss-Prot database for homologous proteins from bacteria, plants, etc, by searching with "enolase". I find ENO2_MAIZE (corn) and ENOA_ANAPL (duck), among others. Note that some enzymes and proteins having similar names may have little or no evolutionary relationship, e.g. polymerase, dehydrogenase. You may have to be more specific, e.g. DNA polymerase, pyruvate dehydrogenase.

1. Hydropathy
1. Copy the accession number for the protein in the PDB file (P00924 for yeast enolase); and write down the numbers for the other two sequences (P26301 and P19140 in my case).
2. Go to "ExPASy", then "Tools", then "ProtScale".
3. Paste your accession number in the space provided
4. Confirm that "Hphob. / Kyte & Doolittle" is selected, and leave the rest as default.
5. "Submit"
6. When ExPASy asks if you want to select a certain portion of the sequence, leave the spaces blank and do the entire sequence. "Submit" again.
7. ExPASy should return the hydropathy plot for a 9-residue averaging window. Identify the maximum (most hydrophobic) and minimum (most hydrophilic).

1. Conservation
1. Go back to the Swiss-Prot entry for your PDB file and copy the whole page, from "ID" through "//".
2. Go to the ClustalW Multiple Sequence Alignment site and paste in the box. Hint: It's easier go back and forth if you open a new browser
3. Go back to one of the Swiss-Prot entries from different kingdoms, copy.
4. Paste immediately below the first sequence in the ClustalW box.
5. Same thing one more time.
6. Step on "Run ClustalW"
7. When the result comes back. Step on the ***.aln file with the right mouse button (hold-down on Macs?) and select "Save Link As..." Save the file to disk (If it's someone else's hard disk, remember to clean up after yourself).
8. Open the saved file in an editor, change the font to 9-point "Courier New", and identify the 9-residue sequence with the most asterisks.
9. Analysis
10. View the PDB file for your protein in RasMol. [preferably as an outside viewer.]
11. Go to the Rasmol command line and select one of your 9-residue segments (my most hydrophilic segment was roughly residues 45-55, so I typed "select 45-55")
12. Color it red ("color red").
13. Do the same with other segments using different colors.
14. Answer the questions.