1. The reactions of disulfide bond formation
Oxidation
SH SH
S S
Reduction
S S SH
Isomerisation
SH S S

2. Thiolates are good nucleophiles Thiols are poor nucleophiles
=> pKa of Cys is critical in disulfide bond formation

3. PDI domain structure a b b’ a’ c x
The minimum requirement to catalyse thiol-disulfide exchange is the a or a’ domain. These have an active site -Trp-Cys-Gly-His-Cys-
b’ domain is involved in substrate binding
b domain function may be primarily structural

4. Activity assay
If your mutant protein has a different activity from the wild-type, either the rate of reaction or KM (or both) for one (or more) of these steps has changed

5. Charge state of active site required for PDI activity-
S SH
pKa of Cys 53 < 5.5
pKa of Cys 56 > 10.0
pKa of “normal” Cysteine thiol is 8.3
Effects step 3 (thiolate needed)
Thiolate needed for step 4 but thiolate reverses step 1

6. Dichotomy
We require Cys56 to be protonated most of the time (high pKa), but at one point in the catalytic cycle, during reoxidation we require it to be deprotonated
i.e. a shift in pKa from a high value to a low value

7. Motion of Arginine 120 side chain into the active site locale modulates the pKa of Cys56

8. PDI Cys pKa dependent Activity assay
If your mutant protein has a different activity from the wild-type, either the rate of reaction or KM (or both) for one (or more) of these steps has changed

9. The reaction of PDI and glutathione
”Mimics” steps 1 and 2 of peptide substrate reaction (GSH replaces peptide substrate).
Both rates depend on [GSH], so difficult to say which step you are observing

10. The reaction of PDI and glutathione
Direct equivalent to steps 3 and 4 of peptide substrate reaction
1st step depends on [GSSG], the 2nd does not, so easy to say which step you are observing

11. Group mutants together: make correlations
Less active
Step 3 effected
Structure changed
Behave like wt
More active
Step 4 effected
Less active
KM effected

12. Group mutants together: make correlations
Explain HOW and WHY the activity is effected
Less active
Step 3 effected
Structure changed
Behave like wt
More active
Step 4 effected
Less active
KM effected

13. For large report:
CD spectra
Denaturation curve
Activity measurement figure
Stopped-flow data table
Then the paper style report for the data being distributed (no structural effects of mutations)
Your construct

14. X Group mutants together: make correlations
Explain HOW and WHY the activity is effected X
Less active
Step 3 effected
Structure changed
Behave like wt
More active
Step 4 effected
Less active
KM effected