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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.
Denaturing agents like urea
disrupt the noncovalent bonds
within the protein that stabilize
Role of β-mercaptoethanol its native tertiary and quaternary
in reducing disulfide bonds: 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 "off-
pathway" 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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