LECTURE 11: PROTEIN FOLDING & PROTEIN-LIGAND INTERACTIONS

Challenge of protein folding

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Nature has the ability of taking a polypetide sequence which has a tremendous amount of freedom and take
it a regularly fold them into a 3-d structreu, which is important for its function
Protein folding are now though in terms of energy landscape
Some proteins still need help, the helpers were initially known as heat-shock proteins
o Scientist observed the expression of these HSPs by exposigin cells to elevated temperature
o To repair that heat damage, the denaturation of protein, the HSPs kicked in
o They are part of the normal cellular machinery
o Chaperones are around whenever proteins are synthesized to make sure theyre folded properly
Three main categories:
o 1. Trigger factor
Sits on ribosomal exit tunnel, where newly synthesized polypeptide sequence is being
released
Plays a passive role, provides a cage, that protects it from extracellular components,
especially other proteins, so they dont interfere
Presence of TF allows 65-80% of newly synthesized polypeptide sequence to fold
properly by themselves
Kepping away other cellular components is sufficient for many newly synthesized
proteins to assum their native states
o 2. DnaK/DnaJ
For polypeptide sequences that are more difficult to fold
Two proteins, think of a snap, a hand
If your arm is the polypeptide sequence that is newly synthesized, Dnak/dnaj literally
wrap around a portion of the polypeptide sequence like a hand
By wrapping around it temporarily, it protects whatever sequence is underneath from the
outside environment
What kind of sequence does it protect?
What stretches do dnak/dnaj preferentially bind to?
What is most vulnerable to undesirable folding?
HYDROPHOBIC REGIONS
o Remember, short stretches of hydrophobic amino acids, which can be
part of a polypeptide sequence, when exposed to the environment can
do 2 things:
1. They can either spontaneously fold/collapse, of which the
driving force is the hydrophobic effect, minimize surface
exposure to aqueous environment
2. Or, worse, if theyre too short to collapse on themselves,
they will find another hydrophobic region to stick together
This is a temporary thing, this binding of dnak/dnaj is facilitated by hydrolysis of ATP
The cell sacrifices a little bit of ATP, that binds to dnak/dnaj, helps it to bind to
hydrophobic regions, once it is hydrolyzed, the dnak/dnaj lets go
Its a short term protection mechanism triggered by ATP
What process requires energy that justifies ATP consumption?
o THE CONFORMATIONAL CHANGE
o For proteins dnak/dnaj to bind, it must undergo a deliberate
conformational change
o Nature uses atp to drive this process
Between dnak/dnaj and TF, 80-90% of all folding processes are taken care of
These two chaperon systems take care of vast majority of folding

3. Chaperonin
Think of a coffee mug, it is a cylindrical assembly, with one side closed and one side
open
Structure:
Two donut shaped structures, bottome and middle portion
On top, there is a little cap
Top down view: it is symmetric, a circle arrangement
Quarternary structure of multiple proteins assembling into a cup shaped
structure
Donut shaped structures are lining of the coffee mug, top is the cap
How does this structure help a protein fold? What happens? How does this chaperonin
work?
It protects it, there is a huge cavity/interior space, but what does it do?
Hint: if you look at the lining of the chaperonin, it is ALL HYDROPHOBIC
RESIDUES
What kind of polypeptide sequences are attracted to the cavity? To dive in to the
cavity?
o HYDROPHOBIC RESIDUES!
What kind of proteins have hydrophobic residues on their surface?
MISFOLDED PROTEINS
The proteins that have made a mistake and have hydrophobic residues on the
surface
The chaperonin is not so much a helper in folding, it is more of a repair system put in
place by the cell to help proteins that have gone down the wrong path
One characteristic of a misfolded proteins is that hydrophobic residues are
exposed, which then tend to aggregate
If there are misfolded proteins they get sucked into the hydrophobic cavity
What happens when a misfolded protein enter the hydrophobic cavity?
Symmetrical nature, 7 subunits that make up each region
In the cap, in the lid region, it is not just to close off, it has 7 identical proteins that each
bind an ATP molecule
The chaperonin, each subunit gets charged with ATP, 7 ATPs
Once a misfolded protein dives into the barrel, the 7 ATPs begin hydrolyzing
What does the hydrolysis of ATP accomplish?
o It releases energy, what is it used for?
o The energy released is used to make the whole molecule twists, the
twist motion, that originates in the cap structure is translated in to the
other 2 donut residues
What does the twist motion do in the donut shaped structures?
The twist motion originates in the cap and extends in to the barrel
The lining of the barrel is hydrophobic
As you twist the hydrophobic residues rotate into the lining of the chaperoning
and are replaced with hydrophilic residues
The twist motion allows the chaperonin, after it has captured the misfolded protein,
attracting it by its hydrophobic lining, it undergoes this conformational change with the
help of ATP hydrolysis and changes the lining to hydrophilic
That switch from hydrophobic to hydrophilic rips the misfolded protein, it forces
it, it unfolds the protein in to an unfolded state
Then the chaperonin lets go
The chaperonin DOESNT HELP FOLD THE PROTEIN, it simply unfolds a misfolded
protein and allows it to try again

Merely a lets try again/second chance for misfolded proteins

It takes a misfolded protein and catapults it back out into an unfolded state, takes
care of the energy barrier
What happens if all this fails?
o 30% of proteins never quite make it despite all of these mechanisms
o What happens with these proteins that dont make it?
Break it down an reuse it
Turns out about 30% of proteins that fold correctly are fed directly into a degradation
machinery
Broken down into individual amino acids
And are reused/recycled
o Its an awful lot of waste, uses up a lot of energy, why does nature choose such a path?
What are the consequences if cell decides not to break it down?
That would make the cell very prone to diseases
A lot of quality control, break down is part of that quality control

Protein structure/protein function

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3 categories:
o 1. Structural role: cytoskeleton structure thats made up of proteins, thats one example, but there
are other examples
Single most obvious structural role of proteins: collagen in tendons, fiber like proteins,
hair and nails
Perming your haird: take polypeptide sequences, break the disulfide bonds that
hold the protein together, shape the hair, and apply a reducing agent
Spider webs, tremendously strong, what makes a spider web so strong? Mostly made up
of polypeptide sequences
o 2. Signaling/binders (ligands)
o Catalysts/enzymes
Signaling binding events:
o How do people visualize the interaction of the protein and its ligand?
The ligand can be another protein or a small molecule
o Emil Fischer:
Proteins have tremendous ability to recognize specific ligands
Ex. Can distinguish between glucose and manose
Enzymes have incredible ability to recognize with very high selectivity
particular small molecules
Explained this phenomenon with lock-and-key
Proteins are like a lock, and ligand is like a key
Lock fits perfectly with the key
Turns out this idea stuck around for 50 years, but as people started understand
more about protein structure and behavior
PROTEINS ARE NOT RIGID, THEY ARE NOT STATIC STRUCTURES,
THEY ARE DYNAMIC, there is motion and conformational changes
o Daniel Koshland:
Mid 50s, came up with induced-fit model
The lock, instead of being conformationally restrained in one orientation, it is, instead,
kind of a fit for the key
As the key slides into the lock, the lock a.k.a the protein undergoes conformational
changes which results in a snug fit
It takes the key to reshape the active site, to make the perfect interaction
Induced-fit in action

Arginine side chains: carry positive charge under physiological conditions

5 Residues all carry positive charge, much positive chagre in protein pocket
o Binding of ATP to binding pocket
o Protein and ligand are not conformationally rigid, wiggle themselves into correct orientation
Everyting is wiggling, not massiveX structural changes, but the system is dynamic, it is
not a static system
o This is how a ligand binding to a protein
In terms of reaction equilibrium you can describe this as a protein and ligand being in
equilibrium with the protein ligand complex
There is not chemical reaction going on, this is simply noncovalent interactions
(hydrophobic interactions, electrostatic, hydrogen bonding) that usually take place in a
protein-ligand interaction
If you look at this system/equilibrium, can you describe how fast the forward reaction
happens? What is the definition of the forward reaction?
How quickly does it form the protein-ligand complex?
It would be concentration of the protein X concentration of the ligand X a rate
constant, ka
o You can do the same thing for the reverse reaction
o Concentration of PL complex X rate constant, kd
o NOTE: small ks are rate constants, capital Ks are equilibrium
constants
If you reach equilibrium, what happens?
By definition, the rate of forward reaction = rate of reverse reaction
(The forward reaction) ka X concentration of protein X concentration of ligand =
kd X concentration of PL complex
This can be rearranged: concentration of product/ concentration of starting
material = Ka
o Capital K so it is an equilibrium constant
o Ka = [PL]/[P][L], Kd = 1/Ka=kd/ka
What is the point of treating this mathematically?
o If you have a protein and two very similar ligands, you ask yourself which of these two ligands
preferentially binds to my protein?
It allows you to quantify this
Protein-ligand binding graph
o X-axis: ligand concentration,
far left, no ligand is present, if there is not ligand present then the fraction of ligandbound protein is 0, theta = 0
As you increase ligand concentration, greater and greater percentage of protein will have
ligand bound to it
As you increase ligand concentration to infinity, it will reach saturation, you will reach
equilibrium
If you use infinitely high concentration of ligand then presumably all of the protein will
be in the ligand-bound form
o Y- axis: fraction of ligand-bound protein: [PL]/([PL]+[P])
What can you learn from these hyperbolic curves?
o You can read the Kd out of the hyperbolic curve
o The concentration of ligand at which 50% of the protein is bound, that ligand concentration is =
the dissociation constant Kd
o The Kd value is a characteristic number for every ligand with a particular protein
The green curve
o It takes a higher ligand concentration to have 50% of the ligand bound
o The Kd is higher
o

Does a higher Kd mean it binds tighter or less tight?

LESS TIGHT
In order to get 50% of the protein bound, you have to use a higher concentration of ligand
A HIGHER Kd MEANS WEAKER BINDING