Download 6. Protein Folding

6. Folding and flexibility
By Assist. Prof. Betul Akcesme
• “Prediction of three dimensional structure of a
protein from its amino acid sequence is the
major unsolved problem in structural
molecular biology”
Introduction
a polypeptide chain acquires its correct 3D structure to achieve the biologically
active native state
Some proteins folds spontaneously,
others require assistance such as enzymes PROTEIN FOLDING
,chaperones)
CHAPERONS
1.Binds to partially folded polypeptide
prevents making unwanted bonding
- promotes the folding
2. After a sequence acquires most of its
correct secondary structure, it has a
looser tertiary structure
3. FINAL native state
MOLTEN GLOBULAR STATE
Molten globular state to native
state spontaneously
How to predict the 3D structure from primary structure?
Major unsolved problem
native state protein is not static:
secondary elements, domains undergo small movements in space:
individual atoms or collectively;
Functional activities of protein : ligand binding trigger
conformation changes.
Globular proteins are only
marginally stable
Denatured state (unfolded)
Native state (folded)
Energy difference: 5 – 15 kcal/mol
(a single H-bond: 2 – 5 kcal/mol)
Two major contributors to the energy difference: Enthalpy and Entropy
Decrease for
spontaneous process
ENTHALPY
energy of noncovalent interactions including the hydrophobic
interactions, hydrogen bonds, and ionic bonds.
These interactions are maximized to produce globular molecule in
native state
Covalent interactions ?
These non‐covalent interactions are therefore stronger and
more frequent in the native state and hence their energy
contribution, enthalpy, is much larger.
The enthalpy difference between native and denatured
states can reach several hundred kcal/mol.
ENTROPY
native state is order, denatured states is disorder –each denatured
structure is unique.
entropy between the native
ordered state and the
denatured state can also reach
several hundred kcal/mol but
in the opposite direction to
the enthalpy difference.
Denatured state
Native state
FREE ENERGY difference between states are very small!
•
Fig.6.22 from Mathews, van Holde & Ahern, Biochemistry, 3rd ed. (2000): Thermodynamics
of protein folding
•
summary of overall interplay between DH and the -TDS components (both hydrophobic effect and
conformational entropy)
• A simple reaction profile for two state reaction. The reaction can be
described in terms entirely analogous to reaction kinetics where G is
the conformational stability of the folded protein (GU − GF). For
folding to occur the change in free energy must be negative
The marginal stability of the native state is biologically
and functionally important!
Globular proteins in correct quantities at appropriate times.
THEREFORE
It is equally important to be able to degrade proteins as it
is to be able to synthesize them.
Globular proteins in living cells usually have a rapid turnover ,which
means their native states is marginally stable.
Some enzymes need structural flexibility, which would be
inconsistent with a rigidly stabilized structure.
Kinetic factors are important for folding
“Calculation for searching all possible conformation in a
random fashion to get the lowest energy is impossible!”
Assuming each peptide has only three (α,β,L) conformation
and time scale is one pico-second (10-12)
(for a polypeptide chain of 150 amino acid residues)
3150 (=1068) possible conformations
Time required is1068 x 10-12 = 1056 seconds (= 1048 years)
cf) actual folding time: 0.1–1000 seconds both in vivo & in
vitro
Levinthal Paradox
To occur on this short time scale, the folding process must be
directed in some way through a “kinetic pathway of unstable
intermediates ” to escape sampling a large number of
irrelevant conformations
Kinetic trapping in a wrong minimum
Kinetic factors are important for folding process it is possible that the
observed folded conformation is not the one with lowest free energy but
rather the most stable of those conformations that are kinetically accessible!
• Protein might be trapped in a local low energy state with a high energy
barrier that prevents it from reaching the GLOBAL ENERGY
MINIMUM. ( give different fold)
• Structure prediction with energy calculation is (might) WRONG!
HOW A LIVING CELL CAN PREVENT THE FOLDING PATHWAY
FROM BECOMING BLOCK AT THE INTERMEDIATE STAGE?
The most common obstacles to correct folding
(1) Aggregation of the intermediates through exposed hydrophobic groups
(2) Formation of incorrect disulfide bonds
(3) Isomerization of proline residues
Can be overcome by CHAPERONES!
Needs “Special proteins that assist the folding protein – chaperone”
• Energy minima in protein
folding.
• Anfinsen’s thermodynamic
hypothesis suggests that the threedimensional structure of a native
protein under physiological
conditions is one in which the
free energy of the system is
the lowest (Anfinsen, C.
B. Science 1973,181, 223–230).
Thus, while proteins may sample
other local minima during
folding, there is only one true
minimum-energy fold.
Alternative way to remove kinetic barrier
• α lytic protease (bacterial enzyme)
• It is synthesized and folded in vivo but not active protein with
prosegment of 77 residues
This segment is excised after folding to PRODUCE ACTIVE ENZYME.
Unfolded α lytic protease without this segment can not
refold easily
ADDING PROSEGMENT can induce refolding
The capacity of folding exits in unfolded enzyme BUT there is a BARRIER present
in folding pathway.
PROSEGMENT REMOVES THIS KINETIC BARRIER by lowering the free energy
of transition state for folding ( like enzyme catalyzes chemical reactions)
Molten globules are intermediates in folding
driving force not very known BUT
• packing of core not formed
•Interior side chains are mobile
•Loops and surface elements remain unfolded
MOLTEN
GLOBULE
UNFOLDED
single native form is
reached, forming native
interactions, including
hydrophobic packing in the
interior & fixing surface
loops
FOLDED
Up to 1 second
Within a few
milliseconds
also some native like
alpha helix and beta
strand positions
Ensemble of conformations are
reduced
Folding occurs through
the progressive
organization of
ensembles of structures
[shown here for the srcSH3 domain (left)] on a
funnel-shaped Free
energy landscape
(right).
Conformational
entropy loss during
folding is compensated
by the free energy
gained as more Native
interactions are formed.
Kinetics is determined by the local roughness of the landscape, relative to
thermal energy. Key interactions in early folding (dashed circle) coincide, for this
protein, with experimentally determined regions.
Burying hydrophobic side chains is a key event
Main mystery of protein folding is…
…The collapse of the unfolded state to generate
the molten globule embodies
• WHAT IS THE DRIVING FORCE BEHIND THE CHOICE OF NATIVE
TERTIARY FOLD FROM A RANDOMLY ORIENTED POLPEPTIDE CAHIN?
1. Secondary structure formation cannot be the
thermodynamic driving force of protein folding…
BECAUSE stable hydrogen bonds can also be formed to water molecules in
the unfolded state. (There is very little change in Free Energy)
2. Large Free energy change by bringing hydrophobic side chains out of contact
with water and into contact with each other in the interior of a globular entity.
Most likely scenario that the polypeptide chain begins to form compact shape
with Hydrophobic side chains partially buried very early in the folding…..
• This scenario has several important consequences
▫ Thus it vastly reduces the number of possible conformations that need
to be searched because only those that are sterically accessible within
this shape can be sampled.
▫ Furthermore, when side chains are buried, their polar backbone –NH and
–CO groups are also buried in a hydrophobic environment, hence unable to
form hydrogen bonds to water –
BUT they bond to each other – so you get alpha and beta
structures.
CONCLUSION
Secondary structure elements NOT DRIVING FORCE for the formation of
Molten Globule
But they are consequences of burying hydrophobic side chains…
Core Questions
• Looking at the protein sequences of globular proteins, one finds that
hydrophobic side chains are usually scattered along the entire sequence,
seemingly randomly.
• In the native state of folded protein, ½ of these side chains are buried, and
the rest are scattered on the surface of the protein, surrounded by
hydrophilic side chains.
• The buried hydrophobic side chains are not clustered in the sequence.
• What causes these residues to be selectively buried during the
early and rapid formation of the molten globule?
• If we wish to solve the folding problem, this must be answered first.
Both single and multiple folding pathways have been
observed
The unfolded state consists of many
rapidly interconverting
conformationally different molecules,
U1...Un.
High energy
unfolded state
Low energy folded
state
The molten globule is an ensemble of
structurally related molecules, M1...Mm,
which are rapidly interconverting and
which slowly change to a single unique
conformation, the folded state F.
The conversion from the molten globule
state to the folded state is slow and passes
through a high energy transition state, T.
Both single and multiple folding pathways have been
observed
• Ui‘s --- unfolded states, many of them.
• Mi’s --- molten globule states. Has most secondary structures, but
less compact.
• Converging to F. During this relatively slower process it passes a
high energy transition state T.
• These facts have been verified by NMR, hydrogen exchange,
spectroscopy, and thermo-chemistry.
•
• Alan Fersht, Cambridge
• The techniques is based on investigation of the effects on the
energetics of folding of single mutation in a protein known
structure
EXAMPLE
Mutation ALA to GLY in solvent exposed face at an
alpha helix ,
the helix is already formed
in the intermediate state
both destabilize both intermediate state and
the native state, as well as transition state
On the other hand, mutations destabilize the
native state but do not affect the energy of the
intermediate or transition state at all
the helix is formed after the
transition state.
The story of barnase
110 amino acids, no disulfide bridges.
3 amino terminal alpha helices, and C terminal 5stranded antiparallel sheet.
Effects of all mutations are examined,
detailed residue characterization : its
folding intermediates and transition states
Folding process:
intermediate molten globule state: already
with most of native secondary
structures, and native like relative
position of alpha helix and beta sheet, as
well as relative positions of beta strands
within the sheet.
Single Major transition state!
One major pathway !
The story of lysozyme
Hen egg-white lysozyme
The native structure has 2 lobes, the first
one with 5 alpha helices, the second is
mainly a 3-stranded antiparallel beta
sheet.
Folding is studied by NMR, Circular dichronism,
fluorescence, hydrogen- deuterium exchanges
Folding process: At 20 milliseconds, two
intermediate states of lysozyme were
detected: one with alpha domain formed, no
beta, the other with neither; in fact a third
(less popular) with both alpha and beta
domains developed.
But is this just because some folds slower?
2 major and one minor
pathways!
The most common obstacles to correct folding
(1) Aggregation of the intermediates through exposed hydrophobic
groups
(2) Formation of incorrect disulfide bonds
(3) Isomerization of proline residues
Can be overcome by CHAPERONES!
Needs “Special proteins that assist the folding protein – chaperone”
• ENZYMES THAT HAVE ROLE IN FOLDING!
Enzymes assists formation of proper disulfide bonds during
folding
• Unfold proteins have no disulfide bonds.
• Formation of disulfide bonds require enzyme to help (oxidation of cysteine
residues).
▫ In bacteria, periplasmic space
 Catalyzed by disulfide bridge forming enzyme (Dsb)
▫ In eukaryotes, disulfide bond formation occurs in E.R. before
transport to cell surface
▫ (not found in cytosol because of reducing environment but IN membrane or
secreted)
 Protein disulfide isomerase(PDI)
 Catalyze internal disulfide exchange to remove folding
intermediates with incorrectly formed disulfide bridge.
Bovine pancreatic trypsin inhibitor (BPTI)
• Thomas Creighton,EMBL
• Peter Kim, MIT
• 58 residues.
• 6 cysteine residues which form 3
disulfide bonds.
• 30-51
• 5-14
• 14-38
• The influence of disulfide bond
formation on folding!
• Folding pathways!
Bovine pancreatic trypsin inhibitor (BPTI)
• Trapping disulfide bonded intermediates as a method for studying
the folding pathways.
First bond randomly form … with 30-51 being more stable, 60%,
Second bond (if the first is 30-51): 5-14, 5-38, 14-38 all possible.
14-38 native, others unfold.
Formation of 5-55 occurs very slowly- buried inside the folded
intermediate and not accessible to oxidizing agents
• Adding the enzyme protein disulfide
isomerase significantly increases the rate of
folding of protein BPTI.
• Prokaryote: Dsb (similar to thioredoxin)
• Eukaryote: PDI (Protein disulfide
isomerase)
• Reduced (SH-containing) PDI catalyzes the rearrangement of a
polypeptide’s non-native disulfide bonds via disulfide interchange
reactions to yield native disulfide bonds.
• (b) Oxidized (disulfide-containing) PDI catalyzes the initial formation of a
polypeptide’s disulfide bonds through the formation of a mixed disulfide.
Reduced PDI can then react with a cellular oxidizing agent to regenerate
oxidized PDI.
Isomerization of proline residues
• Trans‐peptide with C=O and –NH groups pointing in opposite directions was
1000 times preferred to the cis‐peptide.(C=O and –NH groups pointing in
same directions)
• However, when the 2ndresidue is a proline the cis conformer is only 4 times
less stable. cis=-proline peotide in many proteins
• Mainly the Pro trans conformer,(few steric collusions) but the cis version is
found in tight turns and are sometimes essential for conformational flexibility.
• In the native protein the cis‐proline arrangements are stabilized by
tertiary structure interactions, but in the unfolded state there is an
equilibrium between cis/trans isomers.
• When refolding occurs, you can get the proline‐peptide bond in the wrong
form. From a kinetic standpoint, cis‐trans proline isomerization is a very
slow process that can prevent the progress of protein folding by trapping one
or more proline residues crucial for folding in the non‐native isomer,
especially when the native protein requires the cis isomer.
• More prolines—more chance. As noted…cis‐trans isomerization is a slow
process and in vitro is often the rate limiting folding step.
•NEED to get things in right conformations!
• Cis –trans isomerization of
proline peptides is a slow
process and in vitro it is
frequently the rate limiting
step in folding for those
molecules that have been
trapped in a folding
intermediate with wrong
isomers.
• In vivo, rates of this process are enhanced by enzymes initially
called peptidyl prolyl isomerases.
• First one found
cyclophilin
• *impacts the rate of cis‐trans isomerization of proline peptides by a factor of
a million over non enzmatic reaction
Proline
containing
tetrapeptide
Invloved in
immunosupression by
inhibiting T cell proliferation
after binding
immunosupressive drugs (
unrelated to their
isomerase activity)
Graft rejection! (involved in
immunosupression)
The active site is on the
outside of beta barrel
1) CAN A PROTEIN FOLD SPONTANEOUSLY TO ITS
NATIVE STATE?
YES !
Anfinsen's dogma (also known
as the thermodynamic
hypothesis) is a postulate in
molecular biology championed
by the Nobel Prize winner
Christian B. Anfinsen.
2) DOES A PROTEIN SEARCH ALL POSSIBLE OUTCOMES TO FIND
THE FINAL CONFORMATION , THE ONE OF LOWEST FREE
ENERGY?
NO!
How the protein reaches this structure is
really the subject of the field of protein
folding, which has a related dogma called
Levinthal's paradox.
The Levinthal paradox states that the number of possible
conformations available to a given protein is astronomically
large, such that even a small protein of 100 residues would
require more time than the universe has existed to explore
all possible conformations and choose the appropriate one,
it would also arguably make computational prediction of
protein structures under the same basis unfeasible if not
impossible.
Anfinsen Experiment
Protein Folding
• Protein folding considers the question of how
the process of protein folding occurs, i. e.
• unfolded
native state.
• Importance:
• – Predict 3D structure from primary sequence
• – Avoid misfolding related to human diseases
• – Design proteins with novel functions
ANFINSEN: AMINO ACID SEQUENCE DETERMINES PROTEIN SHAPE
The dogma states that, at least for small globular proteins, the native
structure is determined only by the protein's amino acid sequence.
At the environmental conditions (temperature, solvent concentration and
composition, etc.) at which folding occurs,
the native structure is a unique, stable and kinetically accessible minimum of
the free energy.
Hypothesis
“protein amino acid sequence determines the final
shape a protein assumes in a water solution”
UNFOLDING RIBONUCLEASE
1st experiment
• Ribonuclease
▫
the hydrolysis of RNA, and its enzymatic activity depends
entirely upon the protein being in a particular shape.
• 124‐amino acid residues. 4 disulfide bridges
• Bonds can be reduced (electrons removed) with high
concentrations of the sulfhydryl reagent bmercaptoethanol
–S—S—becomes –SHHS—
• Altering the polar nature of the solvent by adding
urea,
▫
the reduced ribonuclease, lacking the disulfide bonds to
resist the stress, open up (denatures) into a random
coil that has no enzyme activity.
1st experiment
When Anfinsen did this, he observed that the ribonuclease protein slowly
regained its enzymatic activity.
• Free of the reducing agent, the sulfhydryl groups (--SH) of the
cysteines were being oxidized by dissolved oxygen from the air, and
the protein was refolding into the catalytically active shape.
• This could only mean that the folding was indeed directed by the
amino acid sequence
Anfinsen Experiment
2nd experiment
• Remove β-mercaptoethanol only,
oxidation of the sulfhydryl group,
then remove urea → scrambled
protein, no activity
• Further addition of trace amounts
3rd
experiment
of β-mercaptoethanol converts the
scrambled form into native form.
• Conclusion: The native form of a
protein has the
thermodynamically most stable
structure.
3rd experiment
1st experiment
2nd experiment
Conclusion
• The original work led Anfinsen to propose his "Thermodynamic
Hypothesis", which states that the native conformation of a
protein is adopted spontaneously.
• In other words, there is sufficient information contained in
the protein sequence to guarantee correct folding from any
of a large number of unfolded states.
• This demonstrated that the shape that a protein realizes in solution
is dictated by amino acid sequence information, which is expressed
in terms of thermodynamic stability
Protein Folding Pathway