Lab 1: PROCEDURE Part 3

PART 3: SURFACE EXPOSURE OF KEY RESIDUES (30mins)

During lab 9, you will use the intrinsic fluorescence properties of tryptophan residues to study the stability of wt and mutant HCAII. The assay relies on the change in fluorescence properties of tryptophan depending on its environment. When a protein is fully folded, most tryptophans are buried; this yields a characteristic spectrum. When a protein is unfolded, all tryptophan residues become exposed, which alters the spectrum. We will use this change in fluorescence upon unfolding to determine whether your mutations affect the stability of HCAII. For now, you will generate some information on the surface exposure of tryptophans. Make sure you save your figures so you can refer back to them during lab 9.

Calculate surface exposure with an online tool

• Download the 2VVA.pdb file from the PDB website (link opens in a new tab): http://www.rcsb.org
• Go to the GETAREA website (link opens in a new tab): http://curie.utmb.edu/getarea.html
• Choose the pdb file you just downloaded. Leave the other fields at the default, enter your email address, and hit “Submit for Analysis.”
• You should now have a read-out for each residue in HCAII and a determination for whether it is inside or outside on the protein. If you scroll to the bottom, you will also get a count of the total number of surface vs. buried atoms.
• Find each tryptophan residue (TRP) and note whether it is listed as in or out.

Visualize surface exposure in PyMOL

• If you closed PyMOL, reopen and open your saved 2VVA.pse file.
• Show all residues as spheres and color gray.
• Select all TRP residues by typing ‘select resn TRP and not name C+O+N+CA’
• Rename the selection trp.
• Color the tryptophans yellow.
• Look at the relative surface locations of each tryptophan. You may want to make the protein transparent (by choosing Setting, Transparency, Spheres) to see where they all are.
• Make a list of each tryptophan with its residue number and whether you think each is completely buried, minimally exposed, significantly exposed, fully exposed.
  • If you like, you can label the tryptophans for easier identification. With the trp residues selected, go to L -> residues
• Take pictures of the trp residues for use in lab 9. You do not have to take separate pictures for each residue – just one or two that allow you to see them all.
• Save this session as 2VVAtrp.pse