Folding Kinetics of Chymotrypsin Inhibitor 2

Jennifer Kuge

MRL Research Experience for Teachers 2007

Mentor: Camille Lawrence

Plaxco Lab- Funded by ICB

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

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

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:

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

+ +

How will the two positive charges near each other affect the folding rate?

WT CI2: 44 s-1

Wild Type CI2

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!

+

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!

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!

+

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!

++

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

+

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!

+

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

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

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

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

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

• 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

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

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

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?

Acknowledgements

• Thank you:– NSF– Camille Lawrence– Martina Michen