Transcript Folding Kinetics of Chymotripsin Inhibitor 2

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
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?
Folding Kinetics
EEaa
kk
k = Ze –Ea/RT
D = unfolded CI2
‡
N = folded CI2
G
‡ = transition state
Ea
Ea
Ea = activation energy
D
N
Reaction coordinate
Wild Type CI2
+
+
WT CI2:
44 s-1
How will the two positive charges near each other affect
the folding rate?
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?
RY48 Mutant
+
WT CI2: 44 s-1
RF48: 1564 s-1
RY48: 2369 s-1
Again, there is only one positive charge. How does
this affect the folding rate?
Faster
than
RF48!
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?
RK48 Mutant
+
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
How will the longer, charged side chain affect the folding
rate?
About RH48 Mutant
the
same as
+
WT!
RH48 at pH 4: 32 s-1
RH48 at pH8: 192 s-1
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
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
RN48 Mutant
+
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
RN48: 30 s-1
How will the smaller, uncharged side chain affect the
folding rate?
Making a Mutant
1. Order primers with 1 amino
acid substitution. (GC, #
m
flanking)
2. Add dNTP, water, primer,
template, enzyme, buffer
3. Thermocycle to make mutant
plasmids
- separate strands
- anneal
- polymerize with primer
(elongate)
m
4. Add Dpn 1 to chew up template
DNA (methylated,
hemimethylated) m
Template
DNA
m
Primers with mutation
m
m
m
m
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
Extracting the C12 Protein
1. Grow 2L of
mutant and spin
down in
centrifuge.
2. Break the E.coli
open by
freezing
3. Add DNAse
spin
Supernatant (-)
Pellet (+)
4. French Press
Pellet (+)
5. Spin down with
French Press/spin
centrifuge into a
pellet. Keep
supernatant
Supernatant (+)
Pellet (-)
6. Add DEAE, then
DEAE/spin
filter
7. FPLC column, gel,
Supernatant (+)
Pellet (-)
pool fractions
8. Dialysis, then filter
Column, dialysis, flash freeze,
lyophilize
9. Flash freeze with
liquid nitrogen
PURIFIED
10. Lyophilize
PROTEIN!
Supernatant
Pellet
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.
Intensity (V)
Varying
[guanidine]
CI2+guanidine
Time (s)
Intensity = c + mx + Ae-kt
*Start with folded CI2
Varying
[guanidine]
Intensity (V)
*Start with unfolded CI2
Unfolding Expt.
CI2
Time (s)
Intensity = c + mx - Ae-kt
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.
Measurement of folding rates: WT CI2
“Chevron plot”
= folding
100
kf
= unfolding
10
1
kobs
0.1
(s-1)
0.01
0
1
2
3
4
0.001
ku
0.0001
GuHCl concentration (M)
5
6
7
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