Download - Folding Kinetics of Chymotrypsin Inhibitor 2
![Page 1: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/1.jpg)
Folding Kinetics of Chymotrypsin Inhibitor 2
Jennifer Kuge
MRL Research Experience for Teachers 2007
Mentor: Camille Lawrence
Plaxco Lab- Funded by ICB
![Page 2: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/2.jpg)
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
![Page 3: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/3.jpg)
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
![Page 4: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/4.jpg)
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:
![Page 5: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/5.jpg)
Ea Ea
Folding Kinetics
D
N
‡
G
Reaction coordinate
k = Ze –Ea/RT
D = unfolded CI2
N = folded CI2
= transition state
Ea = activation energy
‡
Ea k
Ea k
![Page 6: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/6.jpg)
+ +
How will the two positive charges near each other affect the folding rate?
WT CI2: 44 s-1
Wild Type CI2
![Page 7: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/7.jpg)
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!
![Page 8: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/8.jpg)
+
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!
![Page 9: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/9.jpg)
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!
+
![Page 10: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/10.jpg)
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!
++
![Page 11: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/11.jpg)
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
+
![Page 12: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/12.jpg)
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!
+
![Page 13: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/13.jpg)
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 MutantPrimers with mutation
m m
m
m
m
m
mm
m m
m
m
m
m
m
![Page 14: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/14.jpg)
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
![Page 15: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/15.jpg)
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
![Page 16: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/16.jpg)
4. French Press5. Spin down with
centrifuge into a pellet. Keep supernatant
6. Add DEAE, then filter
7. FPLC column, gel, pool fractions
8. Dialysis, then filter9. Flash freeze with
liquid nitrogen10. Lyophilize
Pellet (+)
Pellet (-)Supernatant (+)
Column, dialysis, flash freeze, lyophilize
DEAE/spin
Supernatant (+) Pellet (-)
PURIFIED PROTEIN!
Pellet
Supernatant
French Press/spin
![Page 17: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/17.jpg)
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+guanidineVarying [guanidine] Varying
[guanidine]CI2
Time (s) Time (s)Inte
nsity
(V
)
Inte
nsity
(V
)
Stopped Flow Fluorimeter
Folding Expt.
*Start with unfolded CI2
Intensity = c + mx + Ae-kt Intensity = c + mx - Ae-kt
![Page 18: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/18.jpg)
• 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 em
u[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.
How to Make a Chevron Plot
![Page 19: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/19.jpg)
0.0001
0.001
0.01
0.1
1
10
100
0 1 2 3 4 5 6 7
GuHCl concentration (M)
kobs
(s-1)
Measurement of folding rates: WT CI2“Chevron plot”
= folding
= unfoldingkf
ku
![Page 20: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/20.jpg)
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
![Page 21: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/21.jpg)
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?
![Page 22: Folding Kinetics of Chymotrypsin Inhibitor 2](https://reader035.vdocuments.us/reader035/viewer/2022081504/568144b9550346895db18126/html5/thumbnails/22.jpg)
Acknowledgements
• Thank you:– NSF– Camille Lawrence– Martina Michen