PROTEIN FOLDING
• Major Question: Precisely how is the onedimensional sequence of a protein
programmed to achieve a definitive threedimensional structure? The transition is from
a random coil to a unique structure.
• There are no definitive answers! There are,
however, several important considerations.
RNAase
►Single Chain of
124 residues.
►Stabilized by
4 disulfide bonds
Ribonuclease Experiments
• Disulfide bonds of RNAase reduced with βmercaptoethanol in 8M urea.
• All activity lost; S-S bridges broken, converted
to –SH.
RESTORATION OF ACTIVITY
• Oxidation allowed
 RNAase activity regained
• Original, native conformation formed, with all
correct S-S bonds
reestablished.
• Probability of correct S-S bonds
by chance, 1%.
CONCLUSIONS TO RNAase STUDIES
• Native conformation of a protein is the state
with the lowest Gibbs free energy.
• Proteins follow unique paths to attain a native
state.
• Primary structure possesses sufficient
information for proper folding.
ENERGETICS OF FOLDING
GENERAL PRINCIPLES OF THERMODYNAMICS
ΔG = ΔH – TΔS
“Goldfish are Hell without Tartar Sauce”
Where: ΔG = free energy, energy available for work
ΔH = enthalpy, a measure of bond formation (-)
or a measure of bond breaking (+)
T = temperature, oK
ΔS = entropy, a measure of increasing chaos (+)
or a measure of decreasing chaos (-)
For a spontaneous process, ΔG must be (-)
Levintal’s Paradox (1968)
Reiteration of Anfinsen’s conclusions
Small protein (100 AA’s)
Each AA can assume 3 positions
Total possible structures = 3100 = 5 x 1047
Examine each structure for 1 x 10-13s
Then total search requires 5 x 1034s
This is 1.6 x 1027 years, a period longer
than the age of the universe!
FOLDING OF GLOBULAR PROTEINS
ΔG = ΔH - TΔS
Since ΔG is (-), bond formation and/or an
increase in disorder must predominate.
Random coil breaks bonds with aqueous solvent (+ΔH),
but forms new bonds internally (-ΔH). The enthalpy term
is thus minimal.
Thus, a (+ΔS) is the deciding factor which occurs as a
result of the loss of structured water surrounding
hydrophobic domains on the random coil.
This type of folding is thus entropy driven.
DATA SUPPORTING ENTROPY AS A
DRIVING FORCE FOR FOLDING
•Heat capacity, Cp, (cal/deg-mole) of protein
solutions:
on denaturation, Cp increases
on refolding, Cp decreases
Thus, the aqueous medium is more structured when
the protein is denatured, and less structured when
the protein is in its native conformation.
•Addition of alcohols and similar reagents to protein
solutions leads to protein denaturation.
FOLDING OF FIBROUS PROTEINS
For fibrous proteins, a “collapse” as
described for globular proteins is
prevented by the prevalence of polar
amino acids seeking an aqueous
environment plus a high content of
amino acids for which large alterations
in dihedral bond angles are difficult.
Folding for these proteins is usually
enthalpy driven.
BOTTOM LINE ON FOLDING
ΔG (folding) = -5 to -15 kcal/mole
For a 100 AA protein, where ΔG = -10,
stabilization/AA = 0.1 kcal/mole.
This is less than random thermal motion, 0.6
kcal/mole!
Significance:
evolution has favored flexibility
native proteins are on the borderline of
denaturation
misfolding is a common occurence
Pathways of folding include
the molten globule state.
This slide shows the
structure of the molten
globule state (a) and the
native, folded state (b) of
cytochrome b562. The
molten globule state is
somewhat larger than the
final native conformation
and is readily detected by
size exclusion
chromatography.
Molecular Chaperones
• Why are chaperones needed if folding is
inherent in the sequence?
– to protect nascent proteins from the
concentrated protein matrix in the cell
– to accelerate slow steps
– to ensure correct folding
• Chaperone proteins were first identified as
"heat-shock proteins" (hsp60 and hsp70)
Protein folding pathways.
(a) Chaperone-independent folding.
(b) Hsp70-assisted protein folding.
(c) Folding assisted by Hsp70 and
chaperonin complexes. The
chaperonin complex in E.coli is
GroES-GroEL. The chaperonin
complex in eukaryotic cells is
known as TRiC (for TCP-1 ring
complex) or CCT (cytosolic
chaperonin-containing TCP-1).
Structure and Function of the GroEL-GroES complex. (a) Space-filling representation and
overall dimensions of GroEL-GroES (top view, left;side view, right). GroES is gold; the top , or
apical, GroEL ring is green, and the bottom GroEL ring is red. (b) Section through the center of
the complex to reveal the central cavity. The GroEL-GroES structure is shown as a Ca Carbon
trace. ADP molecules bound to GroEL are shown as space-filling models. (c) Model of the
GroEL cylinder (blue) in action. An unfolded (U) or partially folded (I) polypeptide binds to
hydrophobic patches on the apical ring of a7-subunits, followed by ATP binding and GroES
(red) association. ATP binding triggers a conformational change that buries the a7-subunit
hydrophobic patches (yellow), releasing the polypeptide into the central activity (“Anfinsen
cage”). After about 15 seconds, ATP hydrolysis takes place, followed by binding of ATP to the
lower a7-subunit ring, which causes release of the protein.
ADDITIONAL FACILITATORS
1. PDI – protein disulphide isomerase
PDI-S + -S – S -
PDI - S – S - + S –
2. PPI – peptidyl prolyl isomerase
catalyzes cis-trans isomerization of
X-P bonds.
Ken Dill’s folding funnel.
A good summary view.
Unfolded structures lie
around the top. As the
protein folds, it falls down the
wall of the energy funnel to
more stable conformations.
The native, folded structure is
at the bottom.
Nature Structural Biol.
4, 10-19 (1997).
GENERATION OF ALZHEIMER PEPTIDE
Amyloid precursor protein
cleaved by two proteases
to generate amyloid beta
protein (A).
PRION PRECURSOR PROTEIN
Protein anchored to membrane of nerve cell.
TRANSFORMATION OF PRION PROTEIN
Advent of  pleated sheet dominance