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

2. 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

3. 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

4. Guiding Questions: 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?

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

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

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

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

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

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

11. About the same as WT! RH48 Mutant + 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 at pH 4: 32 s-1 RH48 at pH8: 192 s-1 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? Histidine pKa= 6.8

12. RN48 Mutant + 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 About the same as WT! How will the smaller, uncharged side chain affect the folding rate?

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

14. 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

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

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

17. Stopped Flow Fluorimeter 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. Folding Expt. *Start with unfolded CI2 Unfolding Expt. *Start with folded CI2 Varying [guanidine] CI2+guanidine Varying [guanidine] CI2 Intensity (V) Intensity (V) Time (s) Time (s) Intensity = c + mx + Ae-kt Intensity = c + mx - Ae-kt

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

19. kf ku Measurement of folding rates: WT CI2“Chevron plot” = folding = unfolding

20. 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

21. 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?

22. Acknowledgements • Thank you: • NSF • Camille Lawrence • Martina Michen