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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.
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?
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:
Tertiary structure of
ribonuclease
Berg et al., Fig. 6-1
Renaturation (refolding)
(regain of activity)
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.
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).
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).