BIOC 460, spring 2008

Lecture 7 Protein Folding

Reading: Berg, Tymoczko & Stryer, 6th ed., Chapter 2, pp. 44-53, 61-62

Key Concepts • Proteins fold spontaneously under physiological conditions. – In the equilibrium between the denatured state (unfolded or partially unfolded) and the native state (folded, biologically functional), under physiological conditions the vast majority of molecules are in the native state. • PRIMARY STRUCTURE DETERMINES TERTIARY (AND QUATERNARY) STRUCTURES. – demonstrated by the fact that many proteins can refold from a more or less "random coil" set of conformations without "instructions" from any other cellular components – All the information for 3-dimensional structure is provided by the amino acid sequence. • Proteins can be unfolded (denatured) in vitro by chemical agents like urea, or extremes of heat or pH, and then refolded (renatured) by diluting out the chemical denaturant, changing the pH, etc. • Proteins fold on a defined pathway (or a small number of alternative pathways); they don't randomly search all possible conformations until they arrive at the most stable (lowest free energy) structure.

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Key Concepts, continued • Proteins that don't (re)fold on their own, without assistance, don't need other "instructions" -- they just need "molecular chaperones" (which are also proteins) to keep them from slipping off the folding pathway or to help 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., the spongiform encephalopathies (human CJD, bovine “mad cow” disease), Alzheimer disease, Parkinson disease, Huntington disease – Diseases involving deposits of misfolded proteins (amyloid deposits) result from aggregation of a specific protein, different for different diseases, that has misfolded and formed cross-beta structures that form higher order structures (protofibrils and fibrils/fibers) that are very stable. – One hypothesis is that cellular degradation apparatus can’t keep up with disposal of the abnormally folded protein.

Learning Objectives • Terminology: denaturation, renaturation. • What is the mathematical relationship between the free energy change for a process (ΔG) and the enthalpy change (ΔH) and the change in entropy (ΔS)? • Under "native" (e.g., physiological) conditions, is the folded form of a protein in a higher or lower free energy state than the unfolded state? • Describe the effects of a) urea and b) β-mercaptoethanol on protein structure. (They're different.) • Explain the bottom line conclusion of Anfinsen's experiments that won him the Nobel Prize. • Briefly explain the term “cross-beta” structure and how a small amount of abnormally folded protein might cause formation of amyloid deposits (fibrous aggregates, plaques and tangles) in the brain in neurodegenerative diseases such as the prion diseases (spongiform encephalopathies), Alzheimer disease, Parkinson disease, and Huntington disease. (You don’t need to know names of specific proteins or detailed schemes for aggregation.) • How might the misfolding and amyloid deposits be related to the function of cellular apparatus for "disposal" of misfolded proteins?

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Protein Folding • Process in which a polypeptide chain goes from a linear chain of amino acids with vast number of more or less random conformations in solution to the native, folded tertiary (and for multichain proteins, quaternary) structure REVIEW 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 and folded 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 bonds might shift equilibrium from folded state to unfolded form of protein.

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AA 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 are usually "native"), protein folds spontaneously (right to left in process below). – ΔGunfolding > 0 under "native" conditions – ΔGfolding < 0 under "native" conditions

Proof that AA sequence determines 3-D structure: Anfinsen’s experiments with Ribonuclease A Tertiary structure of ribonuclease Berg et al., Fig. 6-1

Amino acid sequence (primary structure) of bovine ribonuclease • Note the 4 disulfide bonds

Berg et al., Fig. 2-56

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urea (denaturing agent) and β-mercaptoethanol (reducing agent to reduce disulfide bonds) Denaturing agents: Reducing agents (donate electrons): e.g., urea (shown), e.g., thiols, such as β-mercaptoethanol guanidinium HCl

β-mercaptoethanol reduces disulfide bonds in proteins. Role of β-mercaptoethanol in reducing disulfide bonds:

Denaturing agents like urea disrupt the noncovalent bonds within the protein that stabilize its native tertiary and quaternary structure.

(Berg et al., Fig. 2-57)

Anfinsen'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 of the 3-D structure even when backbone is unfolded. • Solution: reducing agent (β-mercaptoethanol) -- reduces disulfide bonds (S–S --> 2 SH groups), so “unfolded” protein is entirely unfolded. Berg et al., Fig. 2-58:

Renaturation (refolding) (regain of activity)

• • • •

Loss of native structure -- denaturation -- inactivates RNase. slow removal of urea (by dialysis) --> refolded protein refolded in absence of reducing agent (so O2 in air could reoxidize SH groups to disulfides) Enzyme refolded and regained activity -- proof the right combinations of S-S bonds had formed (i.e., structure was correct)

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Conclusions from Anfinsen’s Experiments • SPECIFIC CONCLUSIONS: – Correct tertiary structure of RNase backbone had returned. – The right SH groups must have been adjacent to each other prior to reoxidation as a result of the backbone refolding correctly, because disulfide bonds formed spontaneously with the right combinations of Cys residues. • MORE GENERAL CONCLUSIONS (Nobel Prize!): – Native structure is the thermodynamically most stable (favored) state for most proteins. – Native tertiary structure is determined by the primary structure (amino acid sequence) of a protein.

• Folding means arriving at the right combinations of Φ and Ψ angles for every residue in the sequence. • Proteins fold on a defined pathway (or a small number of alternative pathways); they don't randomly search all possible conformations until they arrive at the most stable (lowest free energy) structure. • The “code” that dictates 3-dimensional structure from AA sequence seems to be redundant and more complex than we can currently understand or predict! •

Proteins that don't (re)fold on their own, without assistance, don't need other "instructions" -- they just need "molecular chaperones" (which are also proteins) to keep them from slipping off the folding pathway or to help them to get back on it. –Some chaperones require "expenditure" of energy currency (hydrolysis of ATP) to carry out their function.

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Many diseases are the result of defects in protein folding. • Examples: – Cystic fibrosis involves misfolding and resulting lack of a protein involved in Cl– transport across membranes. – Many neurodegenerative disorders involve abnormal protein aggregation. • Prion diseases (e.g., CJD, Creutzfeldt-Jakob disease) = spongiform encephalopathies (also includes “mad cow” disease, chronic wasting disease in elk and deer, scrapie in sheep, etc.) • Alzheimer disease • Parkinson Disease • Huntington Disease • Partly folded or misfolded polypeptides or fragments may sometimes associate with similar chains to form aggregates. • Partial unfolding of correctly folded proteins may also lead to aggregation. • Aggregates vary in size from soluble dimers and trimers up to insoluble fibrillar structures (amyloid). • Unlike most correctly folded proteins, both soluble and insoluble aggregates can be toxic to cells through unknown mechanisms.

Cystic Fibrosis • defective protein = CFTR (Cystic Fibrosis Transmembrane conductance Regulator) • LACK of normal protein, not the abnormal protein itself, causes disease, so disease-causing mutations are recessive. • Normal protein is a membrane protein (an ATP-regulated chloride channel) in plasma membranes of epithelial cells, that pumps Cl– ions out of cells • Defective (mutant) protein doesn’t fold properly. • Folding intermediates don't dissociate from chaperones, preventing CFTR from insertion into membrane. • When only defective protein (homozygous recessive) is present, Cl– ions accumulate in cells. • High intracellular Cl– concentration makes cells take up H2O from surrounding mucus by osmosis. • Thick mucus accumulates in lungs and other tissues, and its presence in lungs causes difficulty breathing and makes affected individuals very subject to infections like pneumonia.

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Human neurodegenerative disorders e.g., Prion Diseases like Creutzfeldt-Jakob Disease (CJD), Alzheimer Disease, Parkinson Disease, Huntington Disease • formation of protein aggregates, amyloid plaques/tangles, lesions in the brain (Other amyloid-forming diseases affect other organs, e.g., liver or heart.) • Fibrils resulting from aggregation of different proteins (different diseases) have common structural feature: central core of β sheets known as a "cross-beta" structure. • (Many "normal" proteins, even myoglobin, can form amyloid fibrils in vitro when exposed to conditions that partly unfold their native structure.) • Small aggregates (dimers, trimers, etc.) may be more toxic to cells than large fibrils -- small aggregates “seed” formation of larger fibrils. • Anything that increases concentration of partially folded/misfolded molecules, e.g., with hydrophobic patches exposed on their surfaces, that can form inappropriate β sheet structure, can increase rate at which aggregates form. • One theory: Cellular "garbage disposal" apparatus for disposing of abnormally folded proteins, with assistance of chaperones, is overwhelmed in these diseases.

Spongiform Encephalopathies e.g., bovine spongiform encephalopathy (BSE, "mad cow disease"), scrapie (sheep), kuru (humans), Creutzfeldt-Jakob disease (CJD, humans), chronic wasting diesase (elk, mule deer) • Fatal, neurodegenerative diseases, with characteristic "holes" appearing in brain ("sponge"-like appearance) • In prion diseases, there's a normal cellular protein (function often unknown, involving different proteins in different prion diseases) that also occurs in an abnormal conformation. • Infectious, but causative agent is an abnormal protein, a "prion" ("proteinaceous infectious only" protein, PrP) (Stanley Prusiner, Nobel Prize in Physiology/Medicine 1997). 1. Transmissible agent: various sized aggregates of a specific protein 2. Aggregates are resistant to treatment by most protein-degrading enzymes. 3. Protein is largely or completely derived from a cellular protein, PrP, (PrPC) normally present in brain. 4. PrPC has a lot of α-helical conformation; abnormal conformation, PrPSC, has much more β conformation, that tends to aggregate with other PRP molecules. • Abnormal protein can be acquired by infection, or by inheritance (dominant), or spontaneously ("sporadic" -- unknown cause).

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(Spongiform Encephalopathies, continued) • A tiny bit of ABNORMALLY folded protein, PrPSC, can aggregate to form “cross-beta” structures, and small aggregates ("nuclei") can shift folding of normal proteins to join the aggregation process and adopt the abnormal conformation (a shift in a protein conformational equilibrium caused by the protein-protein interactions). • In a sort of cascading "domino effect", a high concentration of abnormally folded protein (PrPSC) forms amyloid plaques (large insoluble fibrous aggregates with β conformation prevalent) in brain. • These cross-beta structures fortunately don't form very fast, but if concentration of the aggregation-prone intermediates increases, rate of cross-beta formation can increase dramatically. Model for prion disease transmission via small aggregates of abnormally folded protein (Berg et al. Fig. 2-61)

(Spongiform Encephalopathies, continued) • Cross-beta structures are in a very thermodynamically stable "offpathway" misfolded state • Hypothesis: misfolded proteins in all kinds of cells are always forming cross-beta structures, but most cell types have adequate degradative machinery to clean up the "garbage" so it doesn't accumulate. Maybe neurons lack adequate molecular machinery to dispose of them.

Alzheimer Disease •

Symptoms: memory loss, dementia, impairment in other forms of cognition and behavior • Not transmissible between individuals • Intracellular aggregates (fibrillar tangles) of protein called tau • Extracellular plaques contain aggregates of β-amyloid peptides (Aβ): 40-42-residue segments derived by proteolytic cleavage of a much larger protein (amyloid precursor protein, APP) attached to plasma membrane of neurons (function unknown) • Peptide has flexible structure, "poised" to form fibrils (ordered peptide aggregates).

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Alzheimer Disease, continued • Anything that increases concentration of Aβ peptides increases probability that a small "protofibril" of misfolded peptide will "seed" (initiate) the aggregation cascade. • The gene for the APP protein is on human chromosome 21. – People with Down Syndrome have 3 copies of chromosome 21 instead of 2. – What might be the consequence of trisomy 21 in terms of probability and age of onset of Alzheimer disease?

Model of cross-beta structure in amyloid fibrils (Berg et al. Fig. 2-62) •extended parallel beta sheet structures (deduced from solid-state NMR studies)

Schematic diagram of amyloid fiber formation by Aβ from cross-beta structures • Aggregation leads to deposition of amyloid (neuritic) plaques in brain. • Protofibrils, microaggregates that are precursors of the much larger plaques, seem to be responsible for neurotoxicity.

from Serpell et al., Biochemistry 39, 13269 (2000)

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Other diseases involving abnormal folding/aggregation of other proteins • Parkinson Disease – Protein that forms fibrils is α-synuclein. – Lesions ("Lewy bodies") form in cytosol of dopaminergic neurons in substantia negra of brain, accompanied by muscle rigidity and resting tremor. • Huntington Disease (a "polyglutamine disease", trinucleotide expansion disorder) – Hereditary: autosomal dominant allele, caused by mutation that's an expansion (abnormal lengthening) of repeated sequence CAGCAGCAG… in gene for the protein huntingtin, encoding polyGln sequences in the protein. – Huntington disease affects about 1 in 10,000 people in general population -- late onset (middle age), but longer triplet expansions seem to cause earlier onset of more rapidly progressing disease (due to the more highly aggregation-prone mutant protein with longer polyGln sequences) – Protein aggregates accumulate in nuclei and cytoplasm of neurons in striatum region of the brain, which is required for coordinated movements, ultimately killing the neurons. – Disease leads to rigidity and dementia, always fatal. – Other trinucleotide expansion diseases, e.g., spinocerebellar ataxias.

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