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Protein Folding. Neha Barve Lecturer Bioinformatics School Of Biotechnoloy, DAVV, Indore. Protein Folding Related Diseases. Review of Thermodynamics. D G Gibbs free energy D H enthalpy D S entropy Cp heat capacity. Some Useful Physical Constants. RT 2.48 kJ/mol
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Protein Folding Neha Barve Lecturer Bioinformatics School Of Biotechnoloy, DAVV, Indore
Review of Thermodynamics • DG Gibbs free energy • DH enthalpy • DS entropy • Cp heat capacity
Some Useful Physical Constants • RT 2.48 kJ/mol • eo 8.854×10−12 C2 N-1 m-2is 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 • DGfoldfor 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).
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. 1 3 2 5 4 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 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 • Some paths are direct (e.g. fast) • 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
Fastest collapse Slowest folding 1016 conformations Slowest collapse Fastest folding 1010 conformations Transition state 103 conformations 80-90% native contacts 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
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. • Hydrophobic collapse. • Formation of local secondary structure • Formation of the molten globule • ‘Freezing out’ of tertiary structure
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. • 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.
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
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.
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]
