BIOC 460, spring 2008LEC 7, Protein Folding 1Lecture 7Protein FoldingReading: Berg, Tymoczko & Stryer, 6th ed., Chapter 2, pp. 44-53, 61-62Key Concepts• Proteins fold spontaneously under physiological conditions.– In the equilibrium between the denatured state (unfolded or partiallyunfolded) and the native state (folded, biologically functional), underphysiological conditions the vast majority of molecules are in thenative state.• PRIMARY STRUCTURE DETERMINES TERTIARY (ANDQUATERNARY) STRUCTURES.– demonstrated by the fact that many proteins can refold from a moreor less "random coil" set of conformations without "instructions" fromany other cellular components– All the information for 3-dimensional structure is provided by theamino acid sequence.• Proteins can be unfolded (denatured) in vitro by chemical agents likeurea, or extremes of heat or pH, and then refolded (renatured) by dilutingout the chemical denaturant, changing the pH, etc.• Proteins fold on a defined pathway (or a small number of alternativepathways); they don't randomly search all possible conformations untilthey arrive at the most stable (lowest free energy) structure.BIOC 460, spring 2008LEC 7, Protein Folding 2Key Concepts, continued• Proteins that don't (re)fold on their own, without assistance, don't needother "instructions" -- they just need "molecular chaperones" (which arealso proteins) to keep them from slipping off the folding pathway or tohelp them to get back on it.– Some chaperones require "expenditure" of energy currency(hydrolysis of ATP) to carry out their function.• Many diseases are the result of defects in protein folding, e.g., thespongiform encephalopathies (human CJD, bovine “mad cow” disease),Alzheimer disease, Parkinson disease, Huntington disease– Diseases involving deposits of misfolded proteins (amyloiddeposits) result from aggregation of a specific protein, different fordifferent diseases, that has misfolded and formed cross-betastructures that form higher order structures (protofibrils andfibrils/fibers) that are very stable.– One hypothesis is that cellular degradation apparatus can’t keep upwith disposal of the abnormally folded protein.Learning Objectives• Terminology: denaturation, renaturation.• What is the mathematical relationship between the free energy change fora process (ΔG) and the enthalpy change (ΔH) and the change in entropy(ΔS)?• Under "native" (e.g., physiological) conditions, is the folded form of aprotein in a higher or lower free energy state than the unfolded state?• Describe the effects of a) urea and b) β-mercaptoethanol on proteinstructure. (They're different.)• Explain the bottom line conclusion of Anfinsen's experiments that won himthe Nobel Prize.• Briefly explain the term “cross-beta” structure and how a small amount ofabnormally folded protein might cause formation of amyloid deposits(fibrous aggregates, plaques and tangles) in the brain inneurodegenerative diseases such as the prion diseases (spongiformencephalopathies), Alzheimer disease, Parkinson disease, and Huntingtondisease. (You don’t need to know names of specific proteins or detailedschemes for aggregation.)• How might the misfolding and amyloid deposits be related to the function ofcellular apparatus for "disposal" of misfolded proteins?BIOC 460, spring 2008LEC 7, Protein Folding 3Protein Folding• Process in which a polypeptide chain goes from a linear chain ofamino acids with vast number of more or less random conformationsin solution to the native, folded tertiary (and for multichain proteins,quaternary) structureREVIEW OF THERMODYNAMICS:• ΔG = ΔH – TΔS ΔG = change in Gibbs free energy–Negative ΔG means decrease in free energy for a process (favorable)–Reaction would go spontaneously in that direction.• ΔH = change in enthalpy–reflects number and kinds of chemical bonds (including noncovalent interactions like salt links, hydrogen bonds, and van der Waals interactions) in reactants and products–MAKING bonds/interactions gives negative ΔH (favorable)• ΔS = change in entropy–increase in disorder gives positive ΔS (favorable)Background:• ΔGfolding (change in free energy) between unfolded structure andfolded structure is SMALL.• ΔGfolding results from many contributions:– enthalpy changes• electrostatic effects (hydrogen bonds, salt bridges)• solvation/desolvation of charged residues• van der Waals interactions• steric factors– entropy change (2 sources):• entropy (hydrophobic effect)• conformational entropy (degrees of freedom, flexibility)• ΔGfolding results from a near balance of opposing large forces.• Small differences in energy are important -- loss of 1 or 2 hydrogen bondsmight shift equilibrium from folded state to unfolded form of protein.BIOC 460, spring 2008LEC 7, Protein Folding 4AA sequence of protein determines 3-dimensional structure• Sequence specifies conformation.• No other information needed for protein to fold to its native,active 3-dimensional structure• Under "native" conditions (physiological conditions areusually "native"), protein folds spontaneously (right to left inprocess below).– ΔGunfolding > 0 under "native" conditions– ΔGfolding < 0 under "native" conditionsProof that AA sequence determines 3-D structure:Anfinsen’s experiments with Ribonuclease AAmino acid sequence (primary structure) of bovine ribonuclease• Note the 4 disulfide bondsBerg et al., Fig. 2-56Tertiary structure of ribonucleaseBerg et al., Fig. 6-1BIOC 460, spring 2008LEC 7, Protein Folding 5urea (denaturing agent) andβ-mercaptoethanol (reducing agent to reduce disulfide bonds)β-mercaptoethanol reduces disulfidebonds in proteins.Reducing agents (donate electrons):e.g., thiols, such as β-mercaptoethanol Role of β-mercaptoethanol in reducing disulfide bonds:(Berg et al., Fig. 2-57)Denaturing agents like urea disrupt the noncovalent bondswithin the protein that stabilizeits native tertiary and quaternarystructure.Denaturing agents:e.g., urea (shown),guanidinium HClAnfinsen's experiments: unfolding and refolding RNase• He unfolded RNase with denaturing agent (8 M urea).• problem: 4 S–S bonds in RNase (covalent crosslinks) "staple in" some ofthe 3-D structure even when backbone is unfolded.• Solution: reducing agent (β-mercaptoethanol) -- reduces disulfidebonds (S–S --> 2 SH groups), so “unfolded” protein is entirely unfolded.• Loss of native structure --
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