Lecture 1
How do proteins fold? Unfolded linear polypeptide ordered protein.
Concept: Rnase-A only fully active when folded into native structure (low denaturant). By
measuring the activity, he determined the extent of the native structure present in the sample.
Connection between the amino acid sequence and the biologically active conformation.
Ways of studying protein folding
- Classical way: Using temperature, detergents (SDS), salt concentration, chaotropic agents
(urea, guanidium hydrochloride), cosmotropic agents, change the Ph. We remove the
denaturant and the protein folds back again. Process happens spontaneously without any
input from the outside. This means the amino acid sequence determines the final structure of
the protein.
How to draw the structure of the protein? Crystallography, NMR, cryo-EM.
This should give us information where the atoms are in space. (Ribbon diagram).
Molecular interactions: Distance matters.
- Covalent: Chemical bond
o Disulfide bonds: Not all proteins have this bond. Stabilize proteins in non-
reducing conditions. Cytosol is reducing. Cytosolic proteins do not have
disulfide bonds.
o Peptide bonds
- Non-covalent: non-chemical bond. Contribute to stability.
o Hydrophobic interactions: Hydrophobic moieties come together. This
is driven by water/ solvent. Water forces hydrophobic molecules to aggregate
to reduce ordered water cages (More entropy is wanted/ disorder).
o Hydrogen bonds: Weaker interaction, not as strong as a
covalent bond.
o Van der Waals interactions: Not permanent dipoles that occur due to
the fluctuating electron clouds around atoms. Weak but everywhere. They
happen when molecules get close enough for these to come into play.
London dispersion force
Van der Waals Repulsion
o Electrostatic interactions: +/-
Ionic interactions (charge-charge): Between fully charged groups (Na+,
Cl-)
Dipole-dipole
Strength van der Waals < hydrogen bonds < ionic interactions < covalent bonds. The strength
of interactions is distance dependent.
Levinthal paradox: if a protein wants to find the lowest energy, it
cannot randomly be done by sampling all possible conformations, it
will take an astronomically long time—longer than the age of the
universe. How do proteins overcome this?
Energy funnel (Folded State = Lowest Energy): Instead of searching randomly, the
protein moves down an energy landscape, where each step reduces the number of
, possibilities. Like a "funnel," misfolded states are eliminated gradually, leading to the
most stable native structure.
Simple protein folding can be described with a 2-state energy landscape.
Transition state: Point in the reaction at which the value of the energy is at a local
maximum. Energy barrier that should be overcame to complete folding. Species in this point
of the reaction are very unstable. Difficult to observe collection of structures formed. If the
protein is in the transition state it has equal chances of folding or unfolding.
- thermodynamic stability is determined by the free energy difference between U and N.
Thermodynamics tells us which state is more favourable.
- folding kinetics are determined by the barrier height (and shape). Kinetics tells us how fast a
process is. The higher the barrier, the slower the reaction rate because fewer molecules have
enough energy to overcome it. A lower activation energy (e.g., with a catalyst/enzyme) means
faster kinetics because more molecules can overcome the barrier.
Second half
Alzheimer: Alzheimer brain, brain shrinks, why? There are aggregates in the brain that destroy
the neurons. This is why people with the disease cannot remember things anymore as neurons
are dying.
Two forms of the protein Tau.
1. Mutant that aggregates, deletion of a lysine. (Picture: Many Tau chambers). Mice
cannot learn.
2. Tau that has mutations but prevent its aggregation. (No Tau chambers). This mouse
can learn.
Chaperones: proteins that assist others to fold properly during or after synthesis, to refold
after partial denaturation. Hsp70, Hsp90, why do they not prevent from getting these
aggregates?
,Basics: Almost every cell has it. There are also specialized chaperones. Chaperones are
conserved because folding happens everywhere (bacteria/GroEL) ((just like ribosomes).
Example (slide 8): Scrambled egg is full of proteins. Entire proteome of the chicken. But what if
we wanted a sunny side up instead starting from a scrambled egg?
We can! Chaperone kit incubated for 20 hours in the presence of ATP; this is stirred.
Chaperones use ATP to change their shape, enabling them to bind and stabilize misfolded
proteins, then release them in a more native form.
SDS-gel shows it looks like the presence of chaperones (+) results in changes in protein bands
over time, potentially indicating disaggregation or refolding of proteins. E. coli Hsp70, E. coli
Hsp100 were used for this experiment. This is very surprising because we used chicken
proteins. Thanks, that this is conserved.
In the case of disease, we have two types of protein aggregates:
- Beta-amyloid plaques (Ab): plaques form outside the cell. They are composed of beta-
amyloid peptides, which are cleaved from the amyloid precursor protein (APP), a
membrane protein. And it gets accumulated outside the cell, this an early phase of the
disease.
- neurofibrillary tangles (Tau?): tangles inside the cell.
You see plaques and tangles. From the aggregation step (plaque) to tangle we have 10 years in
between. We do not understand how these aggregates kill cells (molecular mechanism).
Can we use the same technique of the scrambled egg here? (slide 12)
Proteins appear in very different states in the cell (slide 13). You can get fibrils or condensates
(liquid form of proteins) (from monomers). Understanding that proteins have many states and
how they control it is crucial. These chaperones take decision whether damaged proteins
(fibrils) should be reactivated or removed. How can we develop compounds that target
aggregation?
Mechanism of action of chaperones:
Hydrophobic core is depicted yellow.
Important concept: 1D structure determines 3D.
And chaperones? Hsp70 binds to the most hydrophobic part (core) of the unfolded or
misfolded protein (fast process/ first aid). The partially unfolded protein moves on to a protein
that binds more to the outside of the protein, less hydrophobic parts, that is Hsp90 (slower
process/ doctor). Hsp90 assists in the final folding steps. Then the protein folds. These two
systems cooperate in protein folding.
Folding is spontaneous: once a protein passes this stage, it is committed to a particular folding
trajectory (minutes/ hours).
, Chaperones put
proteins in a state
that they can fold.
Proteins partially fold on their own: Not all unfolded proteins can reach the native (functional)
state without assistance. Majority of proteins (95%) take a misfolding pathway and become
trapped in non-functional or aggregated states. Most proteins require chaperones like Hsp70
or Hsp90 to prevent aggregation and help reach the native state. Chaperones place proteins in
a self-folding state.
Molecular level of Tau:
Intrinsically disordered protein does not adopt any regular structure. This protein does not
have folded part, it is entirely disordered. Normally, it does not aggregate. If we purify it, we
heat it, or boil it Tau won’t aggregate (…).
You need to do something specific to do aggregation, the region called repeat region forms
aggregates. The repeat region has 4 pseudo-repeats. They also have stretches which are
aggregation prone. Red box, aggregation-prone stretches.
Tau is not dramatically hydrophobic, very charged protein. Native Tau is hydrophilic.
- Hydrophobic (non-polar)
- Hydrophilic (polar, charged, and uncharged)
Tau does not adopt a single stable conformation but exists as a dynamic ensemble of
structures.
This repeat region was overexpressed in the mice brain and caused massive formation of
tangles. Experiment: Place mouse on swimming pool and test whether he can find a platform.
1. Pro-aggregating Tau: Mouse cannot learn. Random pathway.
a. What if you stop the overproduction of the protein and if you do the same
test? The mice will learn again and finds the platform. Mice has neurons that
How do proteins fold? Unfolded linear polypeptide ordered protein.
Concept: Rnase-A only fully active when folded into native structure (low denaturant). By
measuring the activity, he determined the extent of the native structure present in the sample.
Connection between the amino acid sequence and the biologically active conformation.
Ways of studying protein folding
- Classical way: Using temperature, detergents (SDS), salt concentration, chaotropic agents
(urea, guanidium hydrochloride), cosmotropic agents, change the Ph. We remove the
denaturant and the protein folds back again. Process happens spontaneously without any
input from the outside. This means the amino acid sequence determines the final structure of
the protein.
How to draw the structure of the protein? Crystallography, NMR, cryo-EM.
This should give us information where the atoms are in space. (Ribbon diagram).
Molecular interactions: Distance matters.
- Covalent: Chemical bond
o Disulfide bonds: Not all proteins have this bond. Stabilize proteins in non-
reducing conditions. Cytosol is reducing. Cytosolic proteins do not have
disulfide bonds.
o Peptide bonds
- Non-covalent: non-chemical bond. Contribute to stability.
o Hydrophobic interactions: Hydrophobic moieties come together. This
is driven by water/ solvent. Water forces hydrophobic molecules to aggregate
to reduce ordered water cages (More entropy is wanted/ disorder).
o Hydrogen bonds: Weaker interaction, not as strong as a
covalent bond.
o Van der Waals interactions: Not permanent dipoles that occur due to
the fluctuating electron clouds around atoms. Weak but everywhere. They
happen when molecules get close enough for these to come into play.
London dispersion force
Van der Waals Repulsion
o Electrostatic interactions: +/-
Ionic interactions (charge-charge): Between fully charged groups (Na+,
Cl-)
Dipole-dipole
Strength van der Waals < hydrogen bonds < ionic interactions < covalent bonds. The strength
of interactions is distance dependent.
Levinthal paradox: if a protein wants to find the lowest energy, it
cannot randomly be done by sampling all possible conformations, it
will take an astronomically long time—longer than the age of the
universe. How do proteins overcome this?
Energy funnel (Folded State = Lowest Energy): Instead of searching randomly, the
protein moves down an energy landscape, where each step reduces the number of
, possibilities. Like a "funnel," misfolded states are eliminated gradually, leading to the
most stable native structure.
Simple protein folding can be described with a 2-state energy landscape.
Transition state: Point in the reaction at which the value of the energy is at a local
maximum. Energy barrier that should be overcame to complete folding. Species in this point
of the reaction are very unstable. Difficult to observe collection of structures formed. If the
protein is in the transition state it has equal chances of folding or unfolding.
- thermodynamic stability is determined by the free energy difference between U and N.
Thermodynamics tells us which state is more favourable.
- folding kinetics are determined by the barrier height (and shape). Kinetics tells us how fast a
process is. The higher the barrier, the slower the reaction rate because fewer molecules have
enough energy to overcome it. A lower activation energy (e.g., with a catalyst/enzyme) means
faster kinetics because more molecules can overcome the barrier.
Second half
Alzheimer: Alzheimer brain, brain shrinks, why? There are aggregates in the brain that destroy
the neurons. This is why people with the disease cannot remember things anymore as neurons
are dying.
Two forms of the protein Tau.
1. Mutant that aggregates, deletion of a lysine. (Picture: Many Tau chambers). Mice
cannot learn.
2. Tau that has mutations but prevent its aggregation. (No Tau chambers). This mouse
can learn.
Chaperones: proteins that assist others to fold properly during or after synthesis, to refold
after partial denaturation. Hsp70, Hsp90, why do they not prevent from getting these
aggregates?
,Basics: Almost every cell has it. There are also specialized chaperones. Chaperones are
conserved because folding happens everywhere (bacteria/GroEL) ((just like ribosomes).
Example (slide 8): Scrambled egg is full of proteins. Entire proteome of the chicken. But what if
we wanted a sunny side up instead starting from a scrambled egg?
We can! Chaperone kit incubated for 20 hours in the presence of ATP; this is stirred.
Chaperones use ATP to change their shape, enabling them to bind and stabilize misfolded
proteins, then release them in a more native form.
SDS-gel shows it looks like the presence of chaperones (+) results in changes in protein bands
over time, potentially indicating disaggregation or refolding of proteins. E. coli Hsp70, E. coli
Hsp100 were used for this experiment. This is very surprising because we used chicken
proteins. Thanks, that this is conserved.
In the case of disease, we have two types of protein aggregates:
- Beta-amyloid plaques (Ab): plaques form outside the cell. They are composed of beta-
amyloid peptides, which are cleaved from the amyloid precursor protein (APP), a
membrane protein. And it gets accumulated outside the cell, this an early phase of the
disease.
- neurofibrillary tangles (Tau?): tangles inside the cell.
You see plaques and tangles. From the aggregation step (plaque) to tangle we have 10 years in
between. We do not understand how these aggregates kill cells (molecular mechanism).
Can we use the same technique of the scrambled egg here? (slide 12)
Proteins appear in very different states in the cell (slide 13). You can get fibrils or condensates
(liquid form of proteins) (from monomers). Understanding that proteins have many states and
how they control it is crucial. These chaperones take decision whether damaged proteins
(fibrils) should be reactivated or removed. How can we develop compounds that target
aggregation?
Mechanism of action of chaperones:
Hydrophobic core is depicted yellow.
Important concept: 1D structure determines 3D.
And chaperones? Hsp70 binds to the most hydrophobic part (core) of the unfolded or
misfolded protein (fast process/ first aid). The partially unfolded protein moves on to a protein
that binds more to the outside of the protein, less hydrophobic parts, that is Hsp90 (slower
process/ doctor). Hsp90 assists in the final folding steps. Then the protein folds. These two
systems cooperate in protein folding.
Folding is spontaneous: once a protein passes this stage, it is committed to a particular folding
trajectory (minutes/ hours).
, Chaperones put
proteins in a state
that they can fold.
Proteins partially fold on their own: Not all unfolded proteins can reach the native (functional)
state without assistance. Majority of proteins (95%) take a misfolding pathway and become
trapped in non-functional or aggregated states. Most proteins require chaperones like Hsp70
or Hsp90 to prevent aggregation and help reach the native state. Chaperones place proteins in
a self-folding state.
Molecular level of Tau:
Intrinsically disordered protein does not adopt any regular structure. This protein does not
have folded part, it is entirely disordered. Normally, it does not aggregate. If we purify it, we
heat it, or boil it Tau won’t aggregate (…).
You need to do something specific to do aggregation, the region called repeat region forms
aggregates. The repeat region has 4 pseudo-repeats. They also have stretches which are
aggregation prone. Red box, aggregation-prone stretches.
Tau is not dramatically hydrophobic, very charged protein. Native Tau is hydrophilic.
- Hydrophobic (non-polar)
- Hydrophilic (polar, charged, and uncharged)
Tau does not adopt a single stable conformation but exists as a dynamic ensemble of
structures.
This repeat region was overexpressed in the mice brain and caused massive formation of
tangles. Experiment: Place mouse on swimming pool and test whether he can find a platform.
1. Pro-aggregating Tau: Mouse cannot learn. Random pathway.
a. What if you stop the overproduction of the protein and if you do the same
test? The mice will learn again and finds the platform. Mice has neurons that
