1 Homework 1 2008 Answers (10 points) Please answer the following questions. Please justify all conclusions. 1. Do the active site cysteines appear oxidized or reduced? The active site cysteines in the structure with added H2O2 are oxidized. The sulfur atoms are 2.1 Å apart, which is the correct distance for disulfide bonds. The electron density for the sulfurs in the 2Fo-Fc map is very good, indicating proper placement. A small positive (green) peak in the Fo-Fc map on the sulfur of Cys 32 indicates the need for more electrons there, which is likely due to incomplete refinement and a slightly high B-factor (B factors are covered later in the course). 2. Does thioredoxin appear to be monomeric or dimeric in the crystal? An argument supporting either answer can be made. It appears that the wild-type protein was disulfide linked to a 2-fold related monomer, and so technically it is dimeric. In the mutant protein, the disulfide is gone but a short beta sheet between molecules suggests a dimer interface exists. The surface area buried is consistent with a weak dimer interaction, but contains an Asp-Asp hydrogen bond, which can only occur at lower pH. Thus, the structure suggests that thioredoxin might be dimeric, particularly at lower pH, but is not conclusive. Solution studies have shown human thioredoxin forms a very weak dimer, somewhat more prominent at lower pH.2 3. How many thioredoxin molecules are in the unit cell? In the asymmetric unit? (Hint: the space group is C2; there is one chain in the PDB file.) There is one molecule in the PDB file and therefore in the asymmetric unit. There are four molecules in the unit cell. Recall that C2 is shorthand for C121, indicating one two-fold symmetry axis. The ‘C’ indicates face centering, generating a second origin and second molecule, and the two-fold symmetry axis generates two more molecules for a total of four. 4. Can you conclude that the Cys 73 mutation was successful? Yes. The mutation was not made in the model and a large negative (red) peak remains on the sulfur atom. Thus there are too many electrons at this site in the model. Careful examination will show that the oxygen of Ser 73 can reside in two positions, one in the original sulfur position, and one rotated to a new position. 5. Describe how you would change Lys 81 in response to the electron density maps. The chi angle at the end of the side chain needs to be changed such that the nitrogen rotates by about 180 degrees, out of the negative (red) Fo-Fc peak surrounding the atom, into the nearby positive (green) Fo-Fc
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