Protein Folding

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Protein Folding

• Neha Barve
• Lecturer
• Bioinformatics
• School Of Biotechnoloy, DAVV, Indore

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Protein Folding Related Diseases
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Review of Thermodynamics

• DG Gibbs free energy
• DH enthalpy
• DS entropy
• Cp heat capacity

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Some Useful Physical Constants

• RT 2.48 kJ/mol
• eo 8.85410-12 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

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Characteristics of a folded protein

• A well defined, generally hydrophobic core
• A generally polar, charged surface
• A unique folding pattern under defined conditions

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Thermodynamics of Protein Folding

• DGfold for most proteins is small, about 5 to
  -20 J/mol lt 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).

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The Experiments of Anfinsen
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What do we mean by folded protein?
Anfinsens 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.
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3
2
5
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These energy barriers are small.
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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 Waals interactions (j/mol)
  stabilizing
• Big effect
• Small effect

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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

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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).

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Electrostatic Contributions

• 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 aas

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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

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Opposing Effects

• Entropy! A folded protein has many fewer
  conformational states than an unfolded one!

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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 foldedconformation.
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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.
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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)

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The Unfolded State
Smaller
Bigger
1030 conformation for 100 residues 1011 years if
folding random
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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.
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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.
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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.
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Energetics and Kinetics
Transition states

1. Some paths are direct (e.g. fast)
2. Some paths must overcome energy barriers (e.g.
   slow)

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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.
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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
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Energetics and Kinetics
Realistic free energy surface from a simple
model. Qo of native contacts C total of
contacts
Dinner et al, TIBS, 2000, 25, 10, p.331
Partial answer to Levinthal Paradox
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More Complex Proteins More Complex Folding
Lysozyme
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70 Rapid collapse
Qa native contacts in a domain Qb native contacts
in b domain
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• Folding proceeds in a fairly regular manner.
• Hydrophobic collapse.
• Formation of local secondary structure
• Formation of the molten globule
• Freezing out of tertiary structure

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The Folding of a HelixFolding_at_home
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Something a Little Bigger
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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
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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.
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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

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Mechanism of Action of Chaperones

• The precise mechanism is not yet known and may
  vary with the substrate. There are two general
  ideas.
• 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.
• 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.

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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.
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Bacterial Folding Complexes
GroES
E. coli Gro EL Apical Equatorial
The thermosome from archaebacteria
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The conformational cycle of GroEL/ES
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Location of the hydrophobic binding sites
(yellow) on the GroEL apical domains in the
GroES-bound 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.
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GroEL-ES in action
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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.
  Thats 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 doesnt 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.

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• 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

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HSP70 AND HSP60 FAMILIES HSP70 AND HSP60 FAMILIES HSP70 AND HSP60 FAMILIES
Location Chaperone Roles
HSP70 Family HSP70 Family HSP70 Family
Prokaryotic cytosol DnaK cofactors DnaJ, GrpE Stabilizes newly synthesised polypeptides and preserves folding competence reactivates heat-denatured proteins controls heat-shock response
Eukaryotic cytosol 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
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HSP60/CHAPERONIN FamilyGroE subfamily HSP60/CHAPERONIN FamilyGroE subfamily 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
TCP-1 subfamily TCP-1 subfamily TCP-1 subfamily
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