protein_folding (Mrs. Neha Barve)

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Protein Folding
Neha Barve
Lecturer
Bioinformatics
School Of Biotechnoloy, DAVV, Indore
Protein Folding Related Diseases
Review of Thermodynamics




DG Gibbs free energy
DH enthalpy
DS entropy
Cp heat capacity
DG  DH  T DS
Some Useful Physical Constants
• RT 2.48 kJ/mol
 eo 8.854×10−12 C2 N-1 m-2 is the vacuum permitivity
 er dielectric constant = scaled vacuum permitivity
– Water 78.5
– Ethanol 24.3
– Benzene 2.27
• Hydrophobicity the transfer of a non-polar solute to an aqueous solvent
accompanied by a large change in Cp
Characteristics of a folded protein
• A well defined, generally hydrophobic core
• A generally polar, charged surface
• A unique folding pattern under defined
conditions
Thermodynamics of Protein Folding
DG  DH  T DS
D G  R T ln K
D G fo ld  D G n a tive  D G d en a tu red
DGfold for most proteins is small, about –5 to -20 J/mol < kT/10/aa
therefore the ratio of unfold/fold varies between 0.13 to 0.0003
• This implies that most proteins are only marginally stable at room temp.
The limited stability comes from a near balance of opposing large forces.
• Small forces can therefore play an important role!
• “Native” state is global minimum of DGfold
• From a strictly thermodynamic point of view, folding is reversible
(Anfinsen, 1973).
The Experiments of Anfinsen
What do we mean by “folded” protein?
Anfinsen’s data suggested that there are two states: folded
and unfolded. We now see the folded state is a collection
of conformers that rapidly interconvert since the energy
barrier is low at room temp. The distribution of the
population in the various conformers depends on the
difference DDGfold according to the Boltzman distribution.
p unfolded
e
 D D G fold / kT
p folded
1
5
4
3
2
These energy barriers are small.
The forces that drive folding
Reversibility implies DGfold is minimized
DG composed of many contributions
• Hydrophobic exclusion (kj/mol) entropic
stabilizing
• Electrostatics: hydrogen bonding and salt bridges (j/mol)
+/-
• entropy (degrees of freedom, flexibility) (kj/mol)
destabilizing
• solvation of polar and charged residues (kj/mol)
stabilizing
• steric repulsion (kj/mol)
destabilizing
• van der Waal’s interactions (j/mol)
stabilizing
Big effect
Small effect
Hydrophobic Effects
• Definition: Transfer of non-polar solutes to an aqueous
solution
• Primary driving force of protein folding!
• Evidence
– 3D structures – cores are mostly hydrophobic residues
 DGfold and DGtransfer (the energy required to transfer a hydrophobic
mol. from an organic solvent to water) similar dependence on T
– Protein stability follows Hofmeister series (SO42-,CH3COO-,Cl-,Br,ClO4-,CNS-) benzene is increasingly soluble in these ions
– Mutation studies
– Computer simulations
Hydrophobic Effects-continued
• At room temp the “hydrophobic effect” is entropic
water molecules form ordered structures around nonpolar
compounds
• Hydrophobic residues collapse in to exclude water
• Additional forces can then stabilize (vdw, h-bond,intrinsic properties)
• Hydrophobic effect is dependent on temperature (unstable at high
AND low temp).
Electrostatic Contributions
Fi 
zi e
2
4 e r r

•
Fi is the attractive/repulsive potential energy between the charges
zi is the unit difference in charge between the 2 ions
•

•
e is the charge of the e- (1.602 × 10-19 C)
eo is the dielectric constant
r is the distance between the 2 ions
•
•
•
•
•
Sensitive to pH and ion concentrations
pH determines total charge (pI)
Ionic strength determines effective range of interactions
Ion pairs contribute 1-3 kcal/mol (on surface)
Ion pairs generally destabilizing if buried (cost up to 19
kcal/mol/ion to completely bury
• Ion pairs contribute ~5-15 kcal/mol per 150 aa’s
Hydrogen Bonds
• ~90% of CO and NH groups H-bonded in a folded protein,
but nearly 100% are H-bonded in an unfolded protein in
water. What are the differences?
• Hydrogen bonds contribute 2-10 kcal/mol
• Destabilizing by themselves (transfer of polar groups from
high to low dielectric medium
• If driven by other forces (e.g. hydrophobic collapse) favour
“internal organization”
Opposing Effects
• Entropy! A folded protein has many fewer
conformational states than an unfolded one!
How many conformations are there in the Native
state?
Simulated folding of a 24 “amino acid”
peptide. The experiment was repeated
many times varying the sequence of the
peptide. Most sequences have one
unique “folded”conformation.
Measuring Unfolding
pH has similar effects.
The Experiments of Anfinsen.
The steep cooperativity of the curves suggest that in solution there are only two states i.e. folded and unfolded.
Kinetics of Protein Folding
• “diffusion model”
• Many parallel pathways operating independently (water
down the mountain)
• Steps are characterized by ensembles instead of unique
conformations
• Modeled by simple chains embedded in a lattice (statistical
mechanics)
The Unfolded State
Smaller
Bigger
1030 conformation for 100 residues
1011 years if folding random
Energetics and Kinetics
DGunfolded
DGfolded
The Levinthal Paradox
The “Golf-course” landscape
All states other than those that are very close to the
folded state are equal in energy. Thus there is no “push”
towards the foled state. This comes directly from the 2
state model of Anfinsen.
Energetics and Kinetics
The Pathway Solution
The first attempt to explain the Levinthal paradox hypothesized
there was a single low energy pathway from unfolded to folded.
Energetics and Kinetics
The Pathway Solution. A slightly more realistic view.
Here essentially all paths lead to folding. Cooperativity comes from
the fact that the energy funnel falls off steeply.
Energetics and Kinetics
Transition states
A) Some paths are direct (e.g. fast)
B) Some paths must overcome energy barriers (e.g. slow)
Structure of the Transition State
The fact that there are many transition states is key since this implies that you can get to the transition state by
many by many different pathways. This is the answer to the Levinthal paradox.
Solution to the Levinthal Paradox
The essence of the solution to the ‘Levinthal Paradox’ that has emerged
from the lattice simulations is that an individual molecule needs to sample only
a very small number of conformations because the nature of the effective
energy surface restricts the search and there are many transition states.
Dinner et al., TIBS, 2000, 25, p.331
Energetics and Kinetics
Fastest collapse
Slowest folding
1016 conformations
Slowest collapse
Fastest folding
Realistic free energy surface from a
simple model.
Qo = # of native contacts
C = total # of contacts
1010 conformations
Transition state
103 conformations
80-90% native contacts
Dinner et al, TIBS, 2000, 25, 10, p.331
Partial answer to Levinthal Paradox
More Complex Proteins = More
Complex Folding: Lysozyme
20%
70%
Rapid collapse
Qa native contacts in a domain
Qb native contacts in b domain
Folding proceeds in a fairly regular manner.
1.
Hydrophobic collapse.
2.
Formation of local secondary structure
3.
Formation of the molten globule
4.
‘Freezing out’ of tertiary structure
The Folding of a Helix
[email protected]
Something a Little Bigger
Real World Example
IgE binding to the IgE receptor in allergy.
Naomi E. Harwood1, James M. McDonnell, Biomedicine and Pharmacotherapy, 2007, v. 61 p. 61
Biologically Assisted Folding: Chaperones
Key point: Chaperones solves the problem of kinetic bottle necks. They essentially unfold a protein when
it gets ‘stuck’ on an unproductive branch of the folding pathway.
Unfolded proteins are dangerous in the cell. Therefore cells have evolved ways to refold proteins – the
chaperones.
Cells have also evolved “quality control” mechanisms that rapidly destroy un- or misfolded proteins in
the endoplasmic reticulum. These latter mechanisms will not be discussed.
Properties of Chaperones
•
Molecular chaperones interact with unfolded or partially folded protein
subunits, e.g. nascent chains emerging from the ribosome, or extended chains
being translocated across subcellular membranes.
• They stabilize non-native conformation and facilitate correct folding of protein
subunits.
• They do not interact with native proteins, nor do they form part of the final
folded structures. They also do not bind natively unfolded proteins.
• Some chaperones are non-specific, and interact with a wide variety of
polypeptide chains, but others are restricted to specific targets.
• They often couple ATP binding/hydrolysis to the folding process.
• Essential for viability, their expression is often increased by cellular stress.
• Main role: They prevent inappropriate association or aggregation of exposed
hydrophobic surfaces and direct their substrates into productive folding,
transport or degradation pathways.
Two Classes
• Constitutive – the chaperonins (GroEL/ES)
• Induced by stress – the HSP
Mechanism of Action of Chaperones
The precise mechanism is not yet known and may vary with the substrate. There are two
general ideas.
1.
The chaperone binds to the unfolded form of the protein and sequesters it within the
central cavity. There it undergoes repeated cycles of binding and unfolding until the
native form is reached.
2.
The chaperone binds to the unfolded or misfolded form of the protein transiently and
acts to further unfold it. The protein is never held within the central cavity. The
protein is continuously unfolded and allowed to restart folding until it attains a native
fold.
The GroEL folding machine
The structure of the chaperonin GroEL (hsp60) Left, a low resolution view of the 14-mer, from the X-ray crystal structure filtered to 25 A resolution. There are 2
contacts (numbered) between the two back-to-back heptameric rings. Right, a single subunit (60 kDa) shown as an alpha-carbon trace. There are three domains,
separated by hinge regions (marked H1 and H2). Bound ATP is shown in space filling form, and the yellow residues are hydrophobic sites of substrate (non-native
polypeptide) binding. These residues are also required for GroES (hsp10) binding, in addition to the blue residues. The charged residues in the inter-ring contacts are
shown in red and blue. The structure was determined by Braig et al (1994) Nature 371, 578-586; Braig et al (1995) Nature Structural Biology 2, 1083-1094.
Bacterial Folding Complexes
GroES
E. coli
Gro EL Apical
Equatorial
The thermosome from archaebacteria
The conformational cycle of
GroEL/ES
Location of the hydrophobic binding sites (yellow) on the GroEL apical domains in the GroESbound ring (top) and open ring (bottom) of GroEL. The large twist of the apical domains in the
bound ring occludes the binding sites so that substrate proteins, originally bound in the open
ring, are ejected from the hydrophobic surface and trapped inside a hydrophilic cavity upon ATP
and GroES binding.
GroEL-ES in action
Take Home Lessons
•
Protein folding is governed by thermodynamics, however the time it takes is
controlled by kinetic limitations.
 DGfold is usually a small negative number. However, it is made up of large,
opposing numbers (entropy and enthalpy) which nearly balance out. That’s
why it is exceptionally difficult to predict DGfold.
• The hydrophobic effect is the most important force driving folding. This is
primarily an enthalpic phenomenon.
• The kinetics of protein folding can be fast because an individual molecule
doesn’t have to sample a large number of conformations.
• Cells have evolved a variety of methods to assist protein folding and protect
against misfolding. This (un)folding machinery is usually ATP dependent.
• The “folded” state of a protein actually consists of a number of rapidly
interconverting, similar conformers.
FAMILIES OF MOLECULAR CHAPERONES
Small heat shock proteins (hsp25) [holders]
• protect against cellular stress
• prevent aggregation in the lens (cataract)
Hsp60 system (cpn60, GroEL) ATPase [(un)folders]
• protein folding
Hsp70 system (DnaK, BiP) ATPase [(un)folders]
• stabilization of extended chains
• membrane translocation
• regulation of the heat shock response
Hsp90 ATPase [holder]
• binding and stabilization/ regulation of steroid receptors, protein kinases
Hsp100 (Clp) ATPase [unfolder]
• thermotolerance, proteolysis, resolubilization of aggregates
Calnexin, calreticulin
• glycoprotein maturation in the ER
• quality control
Folding catalysts: PDI, PPI [folders]
Location
HSP70 AND HSP60 FAMILIES
Chaperone
Roles
HSP70 Family
Prokaryotic cytosol
DnaK cofactors DnaJ, GrpE
Stabilizes newly synthesised
polypeptides and preserves
folding competence;
reactivates heat-denatured
proteins; controls heat-shock
response
SSA1, SSB1(yeast) Hsc/hsp70,
hsp40
(mammalian)
Protein transport across organelle
membranes; binds nascent
polypeptides; dissociates
clathrin from coated
vesicles; promotes
lysosomal degradation of
cytosolic proteins
ER
KAR2, BiP/Grp78
Protein translocation into ER
Mitochondria/ Chloroplasts
SSC1, ctHsp70
Protein translocation into
mitochondria; Insertion of
light-harvesting complex
into thylakoid membrane
Eukaryotic cytosol
HSP60/CHAPERONIN FamilyGroE subfamily
Prokaryotic cytosol
GroEL/ GroES
Protein folding, including
elongation factor, RNA
polymerase. Required for phage
assembly
Mitochondria/ Chloroplasts
Hsp60/10 Cpn60/10
Folding and assembly of
imported proteins
Archaebacterial cytosol
TF55 Thermosome
Binds heat-denatured proteins
and prevents aggregation
Eukaryotic cytosol
TCP-1, CCT, or Tric
Folding of actin and tubulin;
folds firefly luciferase in vitro
TCP-1 subfamily
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