Content 2: Chapter 6: Folding & Flexibility
Hydrophobic interaction stabilize protein structure
• Globular proteins:
- hydrophilic (polar or charged) amino acids outside, water exposed
- hydrophobic (apolar) amino acids packed together in the middle
• β-α-β motif
- hydrophilic aa at top of the helix (slovents exposed)
- hydrophobic aa packed against hydrophobic aa of the β-sheets (shielded
from water molecules)
‘Hydrophobic Effect’
• Why do hydrophobic aa assemble together?
• Hydrophobic species break the hydrogen network in water
-> energetically unfavoured!
• Assembly of hydrophobic species in solution minimizes the damage to the hydrogen
network
Active protein conformation
• Active conformation of proteins is their “native state”
• Unfolded, denatured state
- high temperature
- high pressure
- high concentration of chaotropic agents (chemicals)
e.g.urea (8 M) or guanidinium hydrochloride (6 M)
• Equilibrium between the two forms:
- We refer to a folded protein as a native state.
- Native: it occurs in nature, it is happening in nature and has a kind of function there.
- In a nutshell: we have a denatured (unfolded) state and a native (folded) state. In
between we can unfold or refold proteins. To unfold we can use high temperature,
pressure or concentration of chemicals.
Folding Energy
• Each protein has a particular energy and a particular flexibility (entropy)
• Each fold is a point in a multidimensional free energy (ΔG) surface (landscape)
• folding: low energy (prefered): more H-bonds, good packing
• tight packing: costs entropy for the protein (water gets more entropy)
→ A Folding State is a equilibrium on ΔG level between folding vs unfolding
,On the y-axis on such a diagram we would have ΔG (Gibbs free energy, which reflects the
amount of energy that you free by folding down into such a minimum, down you have the
absolute minimum).
X-axis: folding states
Z-axis: folding states
Folded State – ‘Native State’
• Native (folded) state = a local minimum on the free energy landscape
Why is there not only one native state?
In general proteins that have functions within the cell have usually different conformations.
All the conformational changes depict different native states of the protein. So a native form
of a protein just means that it is one of the forms where the protein has its function.
- Folded state of a protein is as soon as it builds up any sort
of structure we call a folded state.
- All the arrows indicate minima intermediate states of the
protein folding.
Q1: Is that the only native state thinkable?
- no, eg. enzymes could have many operational conformations
,Q2: There are many local minima. What are they then?
- folding intermediates, misfolded proteins, various types of aggregates
Q3: What are the characteristics of the global minimum?
- only one, hard to get out
- mostly dead-packed and non-functional -> exceptions?
Molten Globule
• First step: hydrophobic collapse
• Molten globule: globular structure
• So fast, that its within the deadtime of most experiments
• Shows most secondary structure elements as native state
• Yet more loose and less compact
• Energetically more important to bury hydrophobic aa than is freed from H-bonds of
secondary structure: Q: Driving force here?
• A folding intermediate in a local minimum on free energy landscape
Levinthal’s Paradoxon
• Many ways to think about how many possible conformations exist.
• Here is one:
100 aa with 3 possibilities in Ramachandran (α, β, L): 3100 = 1047 conformations possible
Assume that an amino acid can be either in a β-sheet, left handed
or right handed α-helix (α, L). An amino acid can be in 3 different
states and these are the folded states.
• Proteins fold in seconds
• Folding Problem of proteins:
- Hard to predict the 3D structure from primary sequence
(because there are too many options)
- Understand the underlying mechanism of the folding
process
Molecular Dynamics Simulations of Peptides
• Number of found (relevant) unfolded structures is much smaller than the number of
possible unfolded structures
, • So the number of possible unfolded structures must be limited somehow.
Folding Helper Proteins
Three major obstacles in the way:
1) Forming of incorrect disulfide bonds
2) Isomerization of Proline
3) Aggregation of intermediates due to exposed hydrophobic patches
Folding Helper Proteins
1) Forming and Breaking of disulfide bridges
- Disulfide bridge forming enzymes: Dsb
- protein disulfide isomerase: PDI
Forming and breaking of disulfide bridges means they can constrain (beperken) certain
conformations for a while.
2) “Isomerization” of proline residues
- Peptidyl prolyl isomerases
Isomerization of proline residues can be done with helping proteins such as isomerases. The
isomerization of proteins can have an effect to first go into that direction of folding and then
to another one.
3) Chaperones
- Heat shock proteins
- GroEL/GroES complex
Chaperones are large complexes in which proteins get their right conformation.
Disulfide Bonds
• Equilibria during the folding process
• Some are essential bottlenecks to reach the correct folding state
• In eukaryotes: ER then transported to be secreted
• Protein Disulfide Isomerase (PDI) enzymes help
Two conformations of Proline
• Peptide bonds are almost always trans (1000:1). More less in the cis form (1) due to the
sterical hindrance of the side chain.
• Proline can adopt cis conformation more likely (4:1) than others.
Hydrophobic interaction stabilize protein structure
• Globular proteins:
- hydrophilic (polar or charged) amino acids outside, water exposed
- hydrophobic (apolar) amino acids packed together in the middle
• β-α-β motif
- hydrophilic aa at top of the helix (slovents exposed)
- hydrophobic aa packed against hydrophobic aa of the β-sheets (shielded
from water molecules)
‘Hydrophobic Effect’
• Why do hydrophobic aa assemble together?
• Hydrophobic species break the hydrogen network in water
-> energetically unfavoured!
• Assembly of hydrophobic species in solution minimizes the damage to the hydrogen
network
Active protein conformation
• Active conformation of proteins is their “native state”
• Unfolded, denatured state
- high temperature
- high pressure
- high concentration of chaotropic agents (chemicals)
e.g.urea (8 M) or guanidinium hydrochloride (6 M)
• Equilibrium between the two forms:
- We refer to a folded protein as a native state.
- Native: it occurs in nature, it is happening in nature and has a kind of function there.
- In a nutshell: we have a denatured (unfolded) state and a native (folded) state. In
between we can unfold or refold proteins. To unfold we can use high temperature,
pressure or concentration of chemicals.
Folding Energy
• Each protein has a particular energy and a particular flexibility (entropy)
• Each fold is a point in a multidimensional free energy (ΔG) surface (landscape)
• folding: low energy (prefered): more H-bonds, good packing
• tight packing: costs entropy for the protein (water gets more entropy)
→ A Folding State is a equilibrium on ΔG level between folding vs unfolding
,On the y-axis on such a diagram we would have ΔG (Gibbs free energy, which reflects the
amount of energy that you free by folding down into such a minimum, down you have the
absolute minimum).
X-axis: folding states
Z-axis: folding states
Folded State – ‘Native State’
• Native (folded) state = a local minimum on the free energy landscape
Why is there not only one native state?
In general proteins that have functions within the cell have usually different conformations.
All the conformational changes depict different native states of the protein. So a native form
of a protein just means that it is one of the forms where the protein has its function.
- Folded state of a protein is as soon as it builds up any sort
of structure we call a folded state.
- All the arrows indicate minima intermediate states of the
protein folding.
Q1: Is that the only native state thinkable?
- no, eg. enzymes could have many operational conformations
,Q2: There are many local minima. What are they then?
- folding intermediates, misfolded proteins, various types of aggregates
Q3: What are the characteristics of the global minimum?
- only one, hard to get out
- mostly dead-packed and non-functional -> exceptions?
Molten Globule
• First step: hydrophobic collapse
• Molten globule: globular structure
• So fast, that its within the deadtime of most experiments
• Shows most secondary structure elements as native state
• Yet more loose and less compact
• Energetically more important to bury hydrophobic aa than is freed from H-bonds of
secondary structure: Q: Driving force here?
• A folding intermediate in a local minimum on free energy landscape
Levinthal’s Paradoxon
• Many ways to think about how many possible conformations exist.
• Here is one:
100 aa with 3 possibilities in Ramachandran (α, β, L): 3100 = 1047 conformations possible
Assume that an amino acid can be either in a β-sheet, left handed
or right handed α-helix (α, L). An amino acid can be in 3 different
states and these are the folded states.
• Proteins fold in seconds
• Folding Problem of proteins:
- Hard to predict the 3D structure from primary sequence
(because there are too many options)
- Understand the underlying mechanism of the folding
process
Molecular Dynamics Simulations of Peptides
• Number of found (relevant) unfolded structures is much smaller than the number of
possible unfolded structures
, • So the number of possible unfolded structures must be limited somehow.
Folding Helper Proteins
Three major obstacles in the way:
1) Forming of incorrect disulfide bonds
2) Isomerization of Proline
3) Aggregation of intermediates due to exposed hydrophobic patches
Folding Helper Proteins
1) Forming and Breaking of disulfide bridges
- Disulfide bridge forming enzymes: Dsb
- protein disulfide isomerase: PDI
Forming and breaking of disulfide bridges means they can constrain (beperken) certain
conformations for a while.
2) “Isomerization” of proline residues
- Peptidyl prolyl isomerases
Isomerization of proline residues can be done with helping proteins such as isomerases. The
isomerization of proteins can have an effect to first go into that direction of folding and then
to another one.
3) Chaperones
- Heat shock proteins
- GroEL/GroES complex
Chaperones are large complexes in which proteins get their right conformation.
Disulfide Bonds
• Equilibria during the folding process
• Some are essential bottlenecks to reach the correct folding state
• In eukaryotes: ER then transported to be secreted
• Protein Disulfide Isomerase (PDI) enzymes help
Two conformations of Proline
• Peptide bonds are almost always trans (1000:1). More less in the cis form (1) due to the
sterical hindrance of the side chain.
• Proline can adopt cis conformation more likely (4:1) than others.
