Protein Stability Notes GRADED A 2021-2022
□ Affinsen Experiment
• protein active states are compact
• urea breaks hydrogen bonding while mercaptoethanol breaks the disulfide bridges
• What did this experiment tell us?
o 1) Structure is required for function.
o 2) Sequence contains all the information needed for protein folding.
• Why do proteins need to fold?
o 1*. Orient chemical groups precisely in 3-D => “activity”
o 2. Protect from degradation
o 3. Prevent aggregation
□ Protein Folding and Stability Central Questions
• "Protein Folding Problem”: how does sequence determine structure?
• “Inverse Folding Problem”: how does a structure limit the sequence that can specify it?
• How do the particular amino acids in the sequence contribute to the stability of a particular fold?
How is the uniqueness of a fold determined by the sequence?
• What is the mechanism of protein folding and how does the amino acid sequence guide the
folding process?
□ Folding Stability and
Thermodynamics
Stability = free energy difference
between F and U states.
Assumptions:
1. There are only 2 states: folded and
unfolded, no intermediate states
2. The process is entirely reversible
***Stabili ty can only be experimentally
measured where these two conditions have been established
***Numerical measures only valid for a given set of conditions (relative measure)
, Physical Properties of States
unfolded folded
size random coil compact
solvent
exposure/ → all surfaces hydrated → 25-45% unfolded surface area
hydration
solubility non-polar residues buried polar residues
poor exposed better
Number of
conformations
: many (~2.5)n one or a few
• main chain many interior: highly restrained
• side chain exterior: less restrained
secondary none 30-90% helix/sheet/turn
structure
polar satisfied by water interior by: other residues
interactions exterior by: water
□ How do you measure stability?
• You “just” measure Kf and calculate, right? How do
you measure Kf?
o measuring degree of “foldedness” using
▪ CD222nm - measures 2° structure content (a-helix)
▪ Trp/Tyr fluorescence (temperature sensitive)
□ Affinsen Experiment
• protein active states are compact
• urea breaks hydrogen bonding while mercaptoethanol breaks the disulfide bridges
• What did this experiment tell us?
o 1) Structure is required for function.
o 2) Sequence contains all the information needed for protein folding.
• Why do proteins need to fold?
o 1*. Orient chemical groups precisely in 3-D => “activity”
o 2. Protect from degradation
o 3. Prevent aggregation
□ Protein Folding and Stability Central Questions
• "Protein Folding Problem”: how does sequence determine structure?
• “Inverse Folding Problem”: how does a structure limit the sequence that can specify it?
• How do the particular amino acids in the sequence contribute to the stability of a particular fold?
How is the uniqueness of a fold determined by the sequence?
• What is the mechanism of protein folding and how does the amino acid sequence guide the
folding process?
□ Folding Stability and
Thermodynamics
Stability = free energy difference
between F and U states.
Assumptions:
1. There are only 2 states: folded and
unfolded, no intermediate states
2. The process is entirely reversible
***Stabili ty can only be experimentally
measured where these two conditions have been established
***Numerical measures only valid for a given set of conditions (relative measure)
, Physical Properties of States
unfolded folded
size random coil compact
solvent
exposure/ → all surfaces hydrated → 25-45% unfolded surface area
hydration
solubility non-polar residues buried polar residues
poor exposed better
Number of
conformations
: many (~2.5)n one or a few
• main chain many interior: highly restrained
• side chain exterior: less restrained
secondary none 30-90% helix/sheet/turn
structure
polar satisfied by water interior by: other residues
interactions exterior by: water
□ How do you measure stability?
• You “just” measure Kf and calculate, right? How do
you measure Kf?
o measuring degree of “foldedness” using
▪ CD222nm - measures 2° structure content (a-helix)
▪ Trp/Tyr fluorescence (temperature sensitive)
