Download Figure 9-1

Reductive denaturation and oxidative renaturation of RNase A
Plausible mechanism for the thiol- or enzyme-catalyzed disulfide
interchange reaction in a protein
protein disulfide isomerase
C-chain needed
to direct proper
disulfide bond
formation
Primary structure of porcine proinsulin
Determinants of Protein Folding
A. helices/sheets predominate in proteins because
they fill space efficiently
B. protein folding is directed mainly by internal residues (protein
folding is driven by hydrophobic forces)
C. protein structures are organized hierarchically
Hierarchical organization of globular proteins
(subdomains)
Determinants of Protein Folding (cont.)
D. protein structures are highly adaptable
E. secondary structure can be context-dependent
“chameleon” sequence:
AWTVEKAFKTF (unfolded
free in solution)
NMR structure of protein GB1
Determinants of Protein Folding (cont.)
F. dependence of protein fold on primary sequence
created by changing 50%
of the 56 residues in GB1
not all residues have equally
important roles in specifying
a specific fold
X-ray structure of Rop protein, a homodimer of aa motifs that
associate to form a 4-helix bundle
The Levinthal Paradox
2n backbone torsions, n-residue protein: ~10n
structures
time to explore all structures: t = 10n/1013 s-1
for a 100-residue protein: t = 1087 s
Conclusion: proteins fold via an ordered pathway or set
of pathways
Experimental Methods to Monitor
Protein Folding
(UV/ VIS/ fluorescence /CD)
A stopped-flow device:
40 ms dead-times
cold denatured proteins / T-jump
molar
extinction
coefficient
UV absorbance spectra of the three aromatic amino acids,
phenylalanine, tryptophan, and tyrosine
eL and eR differ (circularly
polarized light); measure ∆e
Circular dichroism (CD) spectra of polypeptides
Pulsed H/D Exchange
X-H + D2O  X-D + HOD
Used to follow the time course of protein folding by 2D NMR
a.
b.
c.
d.
e.
Denatured protein in D2O
Dilute with H2O and allow to fold for time tf
Increase pH to initiate D-H exchange (10-40 ms)
Lower pH; allow to completely fold
Determine which amide protons are protonated and deuterated
Landscape Theory of Protein Folding
Polypeptides fold via a series of conformational
adjustments that reduce their free energy and entropy
until the native state is reached.
There is no single pathway or closely related set
of pathways that a polypeptide must follow in folding
to its native state.
The sequence information specifying a particular
fold is both distributed throughout the polypeptide
chain and highly overdetermined.
Folding funnels: An idealized funnel landscape
Folding funnels: The Levinthal “golf course” landscape
Folding funnels: Classic folding landscape
Closer mimic
of an actual
folding
pathway
Folding funnels: Rugged energy surface
Folds via an ordered
pathway: involves well
defined intermediates
Polypeptide backbone and disulfide bonds of native BPTI
(58 residues, three disulfide bonds)
Renaturation of BPTI: protein primary structures evolved to
specify efficient folding pathways as well as stable native
conformations
Folding accessory proteins
A. Protein disulfide isomerases (PDI)
B. Peptidyl prolyl cis-trans isomerases
C. Molecular chaperones
A folding accessory
protein
Reactions catalyzed by protein disulfide isomerase (PDI). (a)
Reduced PDI catalyzes the rearrangement of the non-native
disulfide bonds.
Reactions catalyzed by protein disulfide isomerase (PDI). (b) The
oxidized PDI-dependent synthesis of disulfide bonds in proteins.
homodimer;
eukaryotic; each
subunit consists of
four domains
Cys 36 and 39 exposed
NMR structure of the a domain of human protein disulfide
isomerase (PDI-a) in its oxidized form. (a) The polypeptide
backbone is shown in ribbon form.
Cys 36 is located
in a hydrophobic
patch
Oxidized PDI-a is
less stable than
reduced PDI-a
NMR structure of the a domain of human protein disulfide
isomerase (PDI-a) in its oxidized form. (b) The molecular
structure as viewed from the bottom.
Peptidyl Prolyl Cis-Trans Isomerases (PPIs)
Xaa-Pro peptide bonds: ~10% cis
PPIs catalyze the otherwise slow interconversion
of Xaa-Pro peptide bonds between their cis and
trans conformations; accelerate the folding of
Pro-containing polypeptides.
Two families: cyclophilins and FKBP12 (based on
known inhibitors)
Unfolded proteins in vivo have a great tendency to
form intramolecular and intermolecular aggregates.
Molecular chaperones prevent/reverse improper
associations, especially in multidomain and
multisubunit proteins.
Function by binding solvent-exposed hydrophobic
surfaces reversibly to promote proper folding
Many chaperones are ATPases.
Classes of Chaperones
A. Heat shock proteins 70: 70 kD monomeric proteins
B. Chaperonins: form large multisubunit cage-like assemblies
C. Hsp90: involved in signal transduction; very abundant
in eukaryotes
D. Nucleoplasmins: acidic nuclear proteins involved in
nucleosome assembly
Chaperonins
GroEL/ES system
14 identical ~60 kD
subunits in two rings;
creates a central
cavity
Electron micrograph-derived 3D image of the Hsp60 (GroEL)
chaperonin from the photosynthetic bacterium Rhodobacter
sphaeroides.
X-ray structure of GroEL. (a) Side view perpendicular
to the 7-fold axis.
X-ray structure of GroEL. (b) Top view along the 7-fold axis.
X-ray structure of GroES as viewed along its 7-fold axis.
cis ring
trans ring
X-ray structure of the GroEL-GroES-(ADP)7 complex.
X-ray structure of the GroEL-GroES-(ADP)7 complex.
bound ADP shown
in cis ring
X-ray structure of the GroEL-GroES-(ADP)7 complex.
apical
intermediate
equatorial
Domain movements in GroEL. (a) Ribbon diagram of a single
subunit of GroEL in the X-ray structure of GroEL alone.
Domain movements in GroEL. (b) A GroEL subunit in the
X-ray structure of GroEL-GroES-(ADP)7.
Domain movements in GroEL. (c) Schematic diagram indicating the
conformational changes in GroEL when it binds GroES.
Apical domain of GroEL in complex with a tight-binding
12-residue polypeptide (SWMTTPWGFLHP).
(a)
(b)
Movements of the polypeptide-binding helices of GroEL.
Reaction cycle of
the GroEL/ES
chaperonin system
in protein folding.
Models for GroEL/ES Action
A. Anfinsen cage model: folding within complex
B. Interative annealing: reversible release of partially
folded intermediates
Refolding of RiBisCO; requires
assistance to reach native state
black: no components
red: released only after
native state is reached
green:
released
after one
turnover
Rate of hydrogen-tritium exchange of tritiated RuBisCO.
exchange with solvent
Schematic diagram of the mechanism of stretch-induced
hydrogen exchange by the GroEL/ES system.
Protein Structure Prediction
Secondary structure
a) Chou-Fasman method
Frequency at which a given aa occurs in an a helix in a set
of protein structures = fa = na/n, where na = number of
amino acid residues of the given type that occur in a
helices, and n = total number of residues of this type in the
protein set
Propensity of a particular aa residue to occur in an a helix =
Pa = fa/<fa>, where <fa> is the average value of fa for all
20 residues
Pa > 1: residue occurs with greater than average frequency
in an a helix
Also applies to b-structure
H = strong former
h = former
I = weak former
i = indifferent
b = breaker
B = strong breaker
Propensities and classifications of amino acid residues for a
helical and b sheet conformations.
B. Reverse turns: Rose method
Occur on the surface of a protein; locations
of minimal hydropathy (exclude helical regions)
Rationale for Observed Propensities
For a-helix: appears related to the amount of side-chain
hydrophobic surface buried in the protein
For Pro: low a propensity caused by strain
For Gly: low a propensity caused by reduced entropy and
lack of hydrophobic stabilization
For Ala: high a propensity caused by lack of a g substituent;
reduced entropic cost; minimal hydrophobic stabilization
Computer-based Secondary Structure Algorithms
Combine three or more methods: accurate to ~75%
Jpred: public domain software
The moderate accuracy is caused by failure to take tertiary
interactions into account (tertiary structure influences
secondary structure).
Secondary structure prediction in adenylate kinase
( N-terminal 24 residues)
Tertiary Structure Prediction
a. comparative or homology modeling
b. fold recognition or threading
c. ab initio methods
Protein
Design
Structures of the second zinc finger motif of Zif268 (DNAbinding protein): X-ray structure.
sequence has
only 6 of the
28 residues
identical to Zif268
(5 are similar)
group of Phe
residues replaces
zinc finger
Structure of de novo designed peptide, FSD-1: NMR structure
(a bba motif; 28 residues)
Comparison of the structures of the second zinc finger motif of
Zif268 and FSD-1: best-fit superpositions of their backbones.
Protein Dynamics
Proteins undergo structural motions that have
functional significance.
Conformational fluctuations (breathing motions) in the
oxygen binding protein, myoglobin.
Classes of Motions
1. atomic fluctuations (10-15-10-11 s; 0.01 - 1Å displacements)
2. collective motions (10-12-10-3 s; 0.01 - 5 Å displacements)
3. triggered conformational changes (10-9 -103 s; 0.5 - 10 Å
displacements)
Techniques: crystallography, NMR, MD
blue = least mobile
red = most mobile
The mobility of the GroEL subunit in the X-ray
structure of GroEL alone.
blue = least mobile
red = most mobile
The mobility of the GroEL subunit in the X-ray
structure of the GroEL-GroES-(ADP)7 complex.
The internal motions of myoglobin as determined by a
molecular dynamics simulation: the Ca backbone and the
heme group.
The internal motions of myoglobin as determined by a
molecular dynamics (MD) simulation: an a helix.
Detecting
infrequent
motions (time
scale of seconds)
Exchange rate of
a particular proton
correlates with the
conformational
mobility of its
surroundings
The hydrogen-tritium “exchange-out” curve for hemoglobin that has
been pre-equilibrated with tritiated water.
Conformational Diseases: Amyloid and Prions
Alzheimer’s disease; transmissible
spongiform encephalopathies (TSEs);
amyloidoses
Common characteristic: formation of amyloid fibrils
The involved proteins assume two different
stable conformations (native and amyloid)
Amyloid fibrils: an electron micrograph of
amyloid fibrils of the protein PrP 27-30.
a
b
Fibrils consist mainly of
b-sheets whose b-strands
are perpendicular to the
fibril axis.
Amyloid fibrils (PrP 27-30): Model (a) and isolated (b)
b sheet.
Amyloidogenic proteins
are mutant forms of
normally occuring proteins
Lysozyme
mutants
occur in familial
visceral
amyloidosis
Superposition of wild-type human lysozyme and its D67H mutant.
Prion Diseases
Evidence that the scrapie agent is a protein: scrapie agent is
inactivated by treatment with diethylpyrocarbonate, which reacts
with His sidechains.
Evidence that the scrapie agent is a protein: scrapie agent is
unaffected by treatment with hydroxylamine, which reacts with
cytosine residues.
Evidence that the scrapie agent is a protein: hydroxylamine
rescues diethylpyrocarbonate-inactivated scrapie reagent.
Prion protein conformations: NMR structure of human prion protein
(PrPC). Note the disordered N-terminal tail residues (dots). PrP
may be a cell-surface signal receptor.
Prion hypothesis: PrPSc
induces the conversion of
PrPC to PrpSc
Conversion may be mediated
by a molecular chaperone.
Prion protein conformations: a plausible model for
the structure of PrPSc (very insoluble)
END
Figure 9-36
(heme).
Molecular formula for iron-protoporphyrin IX
Figure 9-37 Primary structures of some representative ctype cytochromes.
Figure 9-38 Three-dimensional structures of the c-type
cytochromes whose primary structures are displayed in Fig.
9-37.
Figure 9-39
rhodanese.
The two-structurally similar domains of