2. Folding Kinetics of Chymotrypsin Inhibitor 2 Jennifer Kuge MRL Research Experience for Teachers 2007 Mentor: Camille Lawrence Plaxco Lab- Funded by ICB

3. Background Information: Proteins are a chain of amino acids Chymotrypsin Inhibitor 2 (CI2) is a small, single domain protein (~80 amino acids) Protease inhibitor found in barley

4. What started this research… Most point mutations: – lead to very little change to the folding rate –slow down the folding rate When Arg48 is changed to Phe48 in CI2, it accelerates the folding rate

5. What feature of a substitution to Phe48 from Arg48 in CI2 contributes to its accelerated folding rate? Unfavorable charge interactions between Arg46 and Arg48 What occurs during the transition state as proteins fold? Guiding Questions:

6. EaEa EaEa Folding Kinetics D N ‡ G Reaction coordinate k = Ze –Ea/RT D = unfolded CI2 N = folded CI2 = transition state E a = activation energy ‡ E a k E a k

7. ++ How will the two positive charges near each other affect the folding rate? WT CI2: 44 s -1 Wild Type CI2

8. RF48 Mutant + Now there is only one positive charge. How does this affect the folding rate? WT CI2: 44 s -1 RF48: 1564 s -1 Faster!

9. + RY48 Mutant Again, there is only one positive charge. How does this affect the folding rate? WT CI2: 44 s -1 RF48: 1564 s -1 RY48: 2369 s -1 Faster than RF48!

10. WT CI2: 44 s -1 RF48: 1564 s -1 RY48: 2369 s -1 RA48: 67 s -1 RA48 Mutant How will the smaller, uncharged side chain affect the folding rate? About the same as WT! +

11. WT CI2: 44 s -1 RF48: 1564 s -1 RY48: 2369 s -1 RA48: 67 s -1 RK48: 25.3 s -1 RK48 Mutant How will the longer, charged side chain affect the folding rate? About the same as WT! + +

12. WT CI2: 44 s -1 RF48: 1564 s -1 RY48: 2369 s -1 RA48: 67 s -1 RK48: 25.3 s -1 RH48: 80 s -1 RH48 Mutant Histidine is mostly charged at a lower pH (pH4). It is mostly uncharged at a higher pH (pH8). What will the folding rate be at pH 6? About the same as WT! RH48 at pH 4: 32 s -1 RH48 at pH8: 192 s -1 Histidine pKa= 6.8 +

13. WT CI2: 44 s -1 RF48: 1564 s -1 RY48: 2369 s -1 RA48: 67 s -1 RK48: 25.3 s -1 RH48: 80 s -1 RN48: 30 s -1 RN48 Mutant How will the smaller, uncharged side chain affect the folding rate? About the same as WT! +

14. 1.Order primers with 1 amino acid substitution. (GC, # flanking) 2.Add dNTP, water, primer, template, enzyme, buffer 3.Thermocycle to make mutant plasmids - separate strands - anneal - polymerize with primer (elongate) 4. Add Dpn 1 to chew up template DNA (methylated, hemimethylated) Template DNA Making a Mutant Primers with mutation m m m m m m m m m m m m m m m

15. 5. Add E.coli to take up DNA 6. Grow on a plate 7. Pick colonies and put into LB+amp media 8. Spin down and send to another lab to be sequenced

16. 1.Grow 2L of mutant and spin down in centrifuge. 2.Break the E.coli open by freezing 3.Add DNAse spin Supernatant (-) Pellet (+) Extracting the C12 Protein

17. 4. French Press 5.Spin down with centrifuge into a pellet. Keep supernatant 6.Add DEAE, then filter 7.FPLC column, gel, pool fractions 8.Dialysis, then filter 9.Flash freeze with liquid nitrogen 10.Lyophilize Pellet (+) Pellet (-) Supernatant (+) Column, dialysis, flash freeze, lyophilize DEAE/spin Supernatant (+)Pellet (-) PURIFIED PROTEIN! Pellet Supernatant French Press/spin

18. Unfolding Expt. * Start with folded CI2 BACKGROUND: Unfolded CI2 fluoresces at 355 nm. Guanidine unfolds CI2. WHAT IT DOES: Mixes 2 solutions and measures the amount of fluorescence emitted by the new mixture over time. WHAT WE USED IT FOR: Finding the observed folding rate of CI2. CI2+guanidine Varying [guanidine] CI2 Time (s) Intensity (V) Stopped Flow Fluorimeter Folding Expt. * Start with unfolded CI2 Intensity = c + mx + Ae -kt Intensity = c + mx - Ae -kt

19. Use stopped flow to collect observed folding rates (k obs ) of the mutant protein at different concentrations of guanidine for both folding and unfolding experiments. Plot the observed folding rates (k obs ) for each concentration of guanidine and fit it to the Chevron plot equation. ln(k obs )= ln(k f e -m f [D] + k u e m u [D] ) m= indicative of the solvent accessible surface area of the protein [D] = concentration of guanidine Folding rate of each mutant (k f ) is found by extrapolating the Chevron plot to zero guanidine. How to Make a Chevron Plot

20. Measurement of folding rates: WT CI2 “ Chevron plot” = folding = unfolding kfkf kuku

21. Conclusion/Next Steps There appears to be a correlation between charge interaction and folding rate. Does CI2 need to have Arg48 in order to inhibit proteases? –Literature shows naturally occuring RW48 and RF48 do not inhibit as well as wild type

22. What did I learn this summer? Research is slow at times Reading what other people have done is important Technique involved One question can lead to another question –Is it beneficial to be more stable? –If so, what is the biological reason for the conservation of this arg48?

23. Acknowledgements Thank you: –NSF –Camille Lawrence –Martina Michen