Lecture 11:Protein Folding & StabilityMargaret A. DaughertyFall 2004How do we go from an unfolded polypeptide chain to acompact folded protein?(Folding of thioredoxin, F. Richards)BIOC 2051). Amino acid sequence dictates structure.2). The native structure represents the lowest energy state for a protein (physiologicalconditions).3). Proteins are densely packed as small organic crystals. Protein Folding: What we know4). A protein cannot sample all possible conformations in finding its native structure (Levinthal’s paradox).5). Protein folding in vitro is a good model for in vivo folding.6). Protein folding is a cooperative process, usually between N <--> U states.7). Intermediates with non-native structure can exist is some protein folding pathways.8). The molten globule is likely to be an intermediate on protein folding pathways.Structure - FunctionProtein FoldingThe Future of Folding9). The protein folding problem will be solved within 5 years (Walter Gilbert, 1988)10). Designed proteins usually turn out to be molten globules.11). We will eventually be able to predict protein structure from sequence. 1). Amino acid sequence dictates structure.2). The native structure represents the lowest energy state for a protein (physiologicalconditions).3). Proteins are densely packed as small organic crystals.4). A protein cannot sample all possible conformations in finding its native structure (Levinthal’s paradox).5). Protein folding in vitro is a good model for in vivo folding.6). Protein folding is a cooperative process, usually between N <--> U states.7). Intermediates with non-native structure can exist is some protein folding pathways.8). The molten globule is likely to be an intermediate on protein folding pathways.BIOC 205Anfinsen’s protein folding experiment1916-1995Ribonuclease A124 aa, pancreatic enzymeBIOC 205Anfinsen’s experiment: sequence dictates structure `57:Nobel Prize in 1972100 % activity0% activity~1% activity (1)(1) a. Reduce protein (β-ME destroys disulfides) b, Unfold protein in urea (2)(2) Remove urea - allow protein to refold Remove β-ME- allow disulfide to reoxidize(3)(3) Remove β-ME - allow disulfides to reoxidize Remove urea - alllow protein to refold(4)(4) Add trace β-ME, warm~10 hrs ==> 100% ACTIVITY Add trace β-ME + cytosolic* fraction~2 minutes ==>100% ACTIVITYBIOC 205Protein foldingKinetic component: A protein cannotsample all possible conformations infinding its native structureThermodynamic component: The nativestructure represents the lowest energystate for a proteinBIOC 205PROTEIN FOLDING IS ENERGETICALLY FAVORABLEFree Energy Favorable(more negative)Unfavorable(more positive)UnfoldedNucleationof secondarystructural elementsInteractionof secondarystructuralelements“molten globule?”NativeBIOC 205Energetic contributions to protein foldingConformational entropyHydrogen bonds*The hydrophobic effect*Electrostatic ForcesOther forces van der Waals ForcesBIOC 205CONFORMATIONAL ENTROPYVS.The polypeptide chain in the unfolded state has more conformationalfreedom than the folded state. Thus, the conformational entropy isa destabilizing component of the native state free energyBoltzmann’s Equation: S = k ln WBIOC 205HYDROPHOBIC EFFECTHydrophobic effect: Thermodynamicconsequence due to avoidance of H20 by theapolar side chains of a protein. Predicted in1959 by Kauzmann (before 3D structures).Major contributor to stabilization ofnative state: proportional to apolar surfacearea buried.BIOC 205ordered H20abouthydrophobesresult inclathratestructuresEntropicallyunfavorableSequestering of hydrophobesto interior of proteinmolecule => Release of H20 isENTROPICALLY FAVORABLEBIOC 205HYDROGEN BONDS IN PROTEINS“Almost allgroups capableof hydrogenbonding are, infact, hydrogenbonded”Baker &Hubbard, 1984BIOC 205Stabilizing force only if protein-protein and water-water H-bondsare more favorable than those of protein-waterH-bond contribution to native state free energy may be small.+H-O-H••••0-HHUnfolded FoldedBIOC 205ELECTROSTATIC FORCESCharge-dipole:Uqm = qmcosθ/εr2Charge-chargeion pairsCharge-dipoleamide-helix dipolehelix-helixion-helix m = dipole momentε = dielectric constantBIOC 205van der Waals Forces: non-polar interactions or dispersionforces: due to local fluctuations in electron densityU = A/r12 - B/r6Repulsive potential dueto bringing two atoms soclose that theirelectron cloudsinterpenetrateAttractive potential due to mutualinduction of electrostatic dipoles.These attractive forces are weak.However, they are additive andcan make a significant contributionto stability when summed over amoleculerBIOC 205Electronicmotion producesan electrostaticattractionFolding Also InvolvesArrangement of S-S bondsBPTI:bovinepancreatictrypsininhibitorBIOC 205Contributions to the native state free energyFor a typical small globular proteins, the favorable and unfavorableinteractions are energetically enormous (~100s kcal/mol) but thedifference in free energy from unfolded to native state:∆Gfold ~ -5 to - 10 kcal/molBIOC 205KINETIC COMPONENT: LEVINTHAL’S PARADOX AND TIME Given an polypeptide sequence, how does it fold into anative conformation in a reasonable amount of time?Example of ribonuclease (124 amino acid residues): if we assume each residue can sample 3 conformations~1050 conformationsIf folding samples a new conformation every 10-13 seconds1030 years to sample all conformationsIn vitro: ribonuclease folds in ~1-2 minutesBIOC 205Single path; one transition stateSingle path; multiple transition statesMultiple paths; onetransition stateMany potential ways for a protein to foldThe native structure of a protein represents its lowest energy stateThe energy landscape of protein folding can be visualized as a funnel inwhich many paths to the lowest energy state, e.g., the folded state arepossible. The paths through the funnel represent kinetic pathways.A. Rapid folding pathwayB. Protein goes through anenergy minimum, which mayslow folding.ENERGY DECREASEunfoldedfoldedBIOC 205entropymolten globulenative stateIS PROTEIN FOLDING IN VITRO A GOOD MODELFOR PROTEIN FOLDING IN VIVO? PARADOXES OF PROTEIN FOLDING:RATES OF CORRECT FOLDING:in vivo: few minutesin vitro: several hoursEFFICIENCY OF CORRECT FOLDING:in vivo: ~ 99%in vitro: a few % for manyOFTEN, in vitro REFOLDING OF POLYPEPTIDE CHAIN DOES NOT RESULT IN NATIVE STRUCTUREBIOC 205ACCESSORY PROTEINS PLAY AN IMPORTANTROLE IN NASCENT PROTEIN