SlideShare a Scribd company logo
• A protein’s increase in energy can be measured
while folding and unfolding
• Measured over time, these measurements can
give kinetic rates of folding and unfolding (kf
and ku)
• We used circular dichroism in a stopped-flow
device to measure L1 kinetics
http://ftp.aip.org/epaps/hfsp_j/E-HJFOA5-2-007807/Bately%20supplementary%20information.pdf
[K]‡=e−ΔG‡/RT
• A chevron plot is a method of presenting protein
kinetics across varying concentrations
• The denaturant, in our case a guanidine
solution, slows protein folding in correlation
with the concentration
http://ftp.aip.org/epaps/hfsp_j/E-HJFOA5-2-007807/Bately%20supplementary%20information.pdf
• A chevron plot is a method of presenting protein
kinetics across varying concentrations
• The denaturant, in our case a guanidine
solution, slows protein folding in correlation
with the concentration
• This denaturation midpoint is where both
reactions are in equilibrium
http://ftp.aip.org/epaps/hfsp_j/E-HJFOA5-2-007807/Bately%20supplementary%20information.pdf
• L1 forms capsomeres which then form virus-like
particles (VLPs)
• We hypothesize that L1 may form capsomeres in
just a few minutes
• The stopped-flow reveals that L1 protein folds
quickly in under 50 milliseconds
http://proteopedia.org/wiki/index.php/Major_capsid_protein_L1
• The mixing chambers create a time
delay
• Residuals from previous mixes can
interfere with data acquisition
• Delays and possible contaminations
were accounted for
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Map%3A_Physic
al_Chemistry_for_the_Biosciences_(Chang)/09%3A_Chemical_Kinetics/9.10%3A_Fast_Reactions_in_Solution
-20
-10
0
10
20
30
40
50
0.009 0.019 0.029 0.039 0.049 0.059 0.069 0.079 0.089 0.099
0.25M Guanidine
Time (s)
Ellipticity
(millidegrees)
n=25
-20
-10
0
10
20
30
40
50
0.004 0.014 0.024 0.034 0.044 0.054 0.064 0.074 0.084 0.094
0.5M Guanidine
Time (s)
Ellipticity
(millidegrees)
n=40
-20
-10
0
10
20
30
40
50
0.009 0.019 0.029 0.039 0.049 0.059 0.069 0.079 0.089 0.099
1.25M Guanidine
Time (s)
Ellipticity
(millidegrees)
n=40
Time (s)
-20
-10
0
10
20
30
40
50
0.008 0.018 0.028 0.038 0.048 0.058 0.068 0.078 0.088 0.098
1.67M Guanidine
Ellipticity
(millidegrees)
n=20
-6
-4
-2
0
2
4
6
8
10
12
14
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
ln(K)
Guanidine Concentraion (M)

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L1 Protein Kinetics

  • 1.
  • 2. • A protein’s increase in energy can be measured while folding and unfolding • Measured over time, these measurements can give kinetic rates of folding and unfolding (kf and ku) • We used circular dichroism in a stopped-flow device to measure L1 kinetics http://ftp.aip.org/epaps/hfsp_j/E-HJFOA5-2-007807/Bately%20supplementary%20information.pdf [K]‡=e−ΔG‡/RT
  • 3. • A chevron plot is a method of presenting protein kinetics across varying concentrations • The denaturant, in our case a guanidine solution, slows protein folding in correlation with the concentration http://ftp.aip.org/epaps/hfsp_j/E-HJFOA5-2-007807/Bately%20supplementary%20information.pdf
  • 4. • A chevron plot is a method of presenting protein kinetics across varying concentrations • The denaturant, in our case a guanidine solution, slows protein folding in correlation with the concentration • This denaturation midpoint is where both reactions are in equilibrium http://ftp.aip.org/epaps/hfsp_j/E-HJFOA5-2-007807/Bately%20supplementary%20information.pdf
  • 5. • L1 forms capsomeres which then form virus-like particles (VLPs) • We hypothesize that L1 may form capsomeres in just a few minutes • The stopped-flow reveals that L1 protein folds quickly in under 50 milliseconds http://proteopedia.org/wiki/index.php/Major_capsid_protein_L1
  • 6. • The mixing chambers create a time delay • Residuals from previous mixes can interfere with data acquisition • Delays and possible contaminations were accounted for https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Map%3A_Physic al_Chemistry_for_the_Biosciences_(Chang)/09%3A_Chemical_Kinetics/9.10%3A_Fast_Reactions_in_Solution
  • 7. -20 -10 0 10 20 30 40 50 0.009 0.019 0.029 0.039 0.049 0.059 0.069 0.079 0.089 0.099 0.25M Guanidine Time (s) Ellipticity (millidegrees) n=25
  • 8. -20 -10 0 10 20 30 40 50 0.004 0.014 0.024 0.034 0.044 0.054 0.064 0.074 0.084 0.094 0.5M Guanidine Time (s) Ellipticity (millidegrees) n=40
  • 9. -20 -10 0 10 20 30 40 50 0.009 0.019 0.029 0.039 0.049 0.059 0.069 0.079 0.089 0.099 1.25M Guanidine Time (s) Ellipticity (millidegrees) n=40
  • 10. Time (s) -20 -10 0 10 20 30 40 50 0.008 0.018 0.028 0.038 0.048 0.058 0.068 0.078 0.088 0.098 1.67M Guanidine Ellipticity (millidegrees) n=20
  • 11. -6 -4 -2 0 2 4 6 8 10 12 14 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 ln(K) Guanidine Concentraion (M)