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Protein
Folding/Unfolding
The Folding Pathway(s)
U
(I ... I)
N
U, unfolded - I, intermediate - N, native
Assembly of proteins from building blocks
Packing of the secondary structure
The major driving force for the folding of proteins
appears to be the burying and clustering of
hydrophobic side chains to minimize their contact
with water: the “hydrophobic effect”.
Structure and Mechanism in Protein Science
A. Fersht (1999)
WH Freeman and Company, New York, USA
Mechanisms for protein folding
The diffusion-collision mechanism.
Some micro-domains are formed prior collapse.
Local elements of native secondary structure are
formed independently of tertiary structure.
The collapse-reorganization mechanism.
NO microdomains formation prior collapse.
Secondary and tertiary structures are formed in
parallel.
A. Fersht (1999): It is unlikely that there is a single mechanism for
protein folding
Folding of an all-beta model protein
The diffusion-collision mechanism
The collapse-reorganization mechanism
Y. Zhou, State University of New York at Buffalo, USA
http://www.smbs.buffalo.edu/phys_bio
The diffusion-collision mechanism
Some microdomains are formed prior collapse
1
20
33
50
60
90
The collapse-reorganization mechanism
NO beta-sheets formation prior collapse
1
20
35
55
70
90
Time course of protein unfolding and refolding
refolding
unfolding
Laser-heating T-jump of a three-helix bundle protein to 25 °C
Experimental data after 800 ns are well fitted to a double exponential
U. Mayor et al. (2003) Nature 421, 863-867
Molecular Dynamics (MD) Simulation
Representative structures from the molecular dynamics simulations.
Snapshots for the transition-state (TS) ensembles identified from the
wild-type (WT) simulations at different temperatures
U. Mayor et al. (2003) Nature 421, 863-867
Molecular Dynamics (MD) Simulation
Structures from the protein 225 °C denaturation simulation shown in
reverse, to illustrate a probable folding pathway of the protein to reach
the native (N) state
U. Mayor et al. (2003) Nature 421, 863-867
Molecular Dynamics (MD) Simulation
The complete folding pathway of a protein
from nanoseconds to microseconds
U. Mayor et al. (2003) Nature 421, 863-867
The “molten globule” states
U
(I ... I)
TS
N
U, Unfolded - I, Intermediate - TS, Transition State - N, Native
The “molten globule” states are partly folded
intermediate states of proteins that are characterized
by having few tertiary interactions, some secondary
structure, and a fluctuating hydrophobic core and by
being separated from the native state by a high
activation energy.
Structure and Mechanism in Protein Science
A. Fersht (1999)
WH Freeman and Company, New York, USA
The structure of a
molten globule.
(A) A molten globule
form of cytochrome
b562 is more open and
less highly ordered than
the native protein,
shown in (B). Note that
the molten globule
contains most of the
secondary structure of
the native form,
although the ends of the
alpha helices are frayed
and one of these helices
is only partly formed.
Molecular Chaperones in the Cytosol
• To become functionally active, newly synthesized protein chains must
fold to unique three-dimensional structures.
• How this is accomplished remains a fundamental problem in biology.
• Although the native fold of a protein is encoded in its amino acid
sequence, protein folding inside cells is not generally a spontaneous
process.
• Many newly synthesized proteins require a complex cellular machinery
of molecular chaperones and the input of metabolic energy to reach their
native states efficiently.
• The various chaperone factors protect nonnative protein chains from
misfolding and aggregation, but do not contribute conformational
information to the folding process.
F.U. Hartl & M. Hayer-Hartl (2002) Science 295, 5561
Mechanisms of accelerated folding
Confinement of non-native protein in the narrow, hydrophilic environment
of the GroEL-GroES cage is suggested to result in a smoothing of the energy
landscape (right), such that formation of certain trapped intermediates is
avoided
F.U. Hartl & M. Hayer-Hartl (2002)
Science 295, 5561
Energy landscapes and modes of function of proteins
(a) The `simplistic' model
of proteins describes an
energy landscape of a
single stable conformer (i)
and a function mode of
either lock and key (ii) or
induced fit (iii). (b) The
`new view' assumes an
ensemble of conformers of
similar free energy (i), and
a mode of function based
on an equilibrium between
two (or more) pre-existing
isomers, only one of which
exerts function (ii).
LC James & DS Tawfik (2003) TIBS 28, 361-368
The co-evolution of fold and function
(a) The enzyme is in equilibrium between different conformations. The native
substrate (yellow) selects the dominant conformer (dark blue). (b) An
alternative conformation potentiates the binding of a second substrate (pink).
(c) Gene duplication enables one copy to evolve improved activity.
LC James & DS Tawfik (2003) TIBS 28, 361-368
Protein folding. A
newly synthesized
protein rapidly attains a
"molten globule" state.
Subsequent folding
occurs more slowly and
by multiple pathways,
some of which reach
dead ends without the
help of a molecular
chaperone. Some
molecules may still fail
to fold correctly; these
are recognized and
degraded by proteolytic
enzymes
Two families of molecular chaperones. The hsp70 proteins act
early, recognizing small patches on a protein's surface. The hsp60like proteins appear to act later and form a container into which
proteins that have still failed to fold are transferred. In both cases
repeated cycles of ATP hydrolysis by the hsp proteins contribute to
a cycle of binding and release of the client protein that helps this
protein to fold.
Molecular Chaperones in the Cytosol
Models for the chaperone-assisted folding of newly synthesized polypeptides.
TF, trigger factor; PFD, prefolding; NAC, nascent chain-associated complex
F.U. Hartl & M. Hayer-Hartl (2002) Science 295, 5561
Molecular Chaperones
in the Cytosol
Chaperones that bind nascent chains:
A) Structures of the ATPase domain and the
peptide-binding domain of Hsp70 shown
representatively for E. coli DnaK.
B) Simplified reaction cycle of the DnaK
system.
C) Structure of archaeal PFD.
F.U. Hartl & M. Hayer-Hartl (2002) Science 295, 5561
The GroEL-GroES
chaperonin system
A) Structure of the complex
B) Simplified reaction of
protein folding in the
GroEL-GroES cage
C) Mechanisms of accelerated
folding
Confinement of non-native
protein in the narrow,
hydrophilic environment of the
GroEL-GroES cage is
suggested to result in a
smoothing of the energy
landscape (right), such that
formation of certain trapped
intermediates is avoided
F.U. Hartl & M. Hayer-Hartl (2002)
Science 295, 5561
Molecular machines
Schnitzer (2001) Nature 410, 878 - 881
Translocation machines - Mitochondria
Once through the Toc complex, the
pathway diverges. On the left is the
pathway for proteins destined for the
matrix. On the right is the pathway for
import of polytopic inner membrane
proteins.
Chap: cytosolic chaperones
70: mitochondrial Hsp70
OM and IM: outer and inner membranes
Outer translocon (Toc), in purple
Inner translocon (Tic), in green
Opening the door to mitochondrial protein import
R.E. Jensen & A.E. Johnson (2001) Nature Struct Biol 8, 1008
Unfolding pathways of barnase
a) during spontaneous unfolding in free-solution
b) during import into mitochondria
The parts of the structure shown in red unfold
early, whereas those shown in blue unfold late.
S. Huang et al. (1999) Nature Struct Biol 6, 1132
Unfolding pathways
of barnase
Barnase is targeted to mitochondrial
membranes by the addition of a
mitochondrial presequence (solid
black bar, lower picture).
Unfolding and import requires an
electrical membrane potential (DY)
across the inner membrane and the
ATP-dependent assistance of
mtHsp70 (hands) in the
mitochondrial matrix
D.N. Hebert (1999) Nature Struct Biol 6, 1084
Translocation machines - Mitochondria
Outer-membrane
proteins with a
complicated
topology pass
through the TOM
complex, then
become integrated
in the membrane
with the assistance
of a separate
sorting and
assembly complex
(SAM).
TOM, Outer translocon
TIM, Inner translocon
Tom20 is the major import receptor
SAM, sorting and assembly complex
Wiedemann et al. (2003) Nature 424, 565-571
Mihara (2003) Nature 424. 505-506
Translocation machines - Chloroplasts
OM and IM: outer and inner membranes
SPP: stromal processing peptidase
C70, 60 and 93: chaperones
Outer translocon (Toc), in green
Inner translocon (Tic), in blue
A GTPase gate for protein import into chloroplasts
F. Kessler & D.J. Schnell (2002) Nature Struct Biol 9, 81-83
Proteasome. Most of the proteins that are degraded in the cytosol
are delivered to large protein complexes called proteasomes. Each
proteasome consists of a central cylinder formed from multiple
distinct proteases. Each end of the cylinder is "stoppered" by a
large protein complex formed from at least 10 types of
polypeptides, some of which hydrolyze ATP.
Toxic proteins in neurodegenerative diseases
The ubiquitin-proteasome system. Proteins targeted for degradation are identified by covalent
linkage to ubiquitin. Selective ubiquitination is accomplished by a series of enzymes (E1, E2, and
E3) that constitute the ubiquitin ligase system. (B) Ubiquitinated substrates are recognized,
unfolded, and degraded in an energy-dependent manner by the proteasome.
JP Taylor et al. (2002) Science 296, 1991
Protein Aggregation
• Large proteins often refold inefficiently, owing to the formation of
partially folded intermediates that tend to aggregate.
• Misfolding originates from interactions between regions of the folding
polypeptide chain that are separate in the native protein. These
nonnative states expose hydrophobic amino acid residues and readily
self-associate into disordered complexes.
• This aggregation process irreversibly removes proteins from their
productive folding pathways, and must be prevented in vivo by
molecular chaperones.
• A certain level of protein aggregation does occur in cells and, in special
cases, can lead to the formation of structured, fibrillar aggregates,
known as amyloid, that are associated with diseases such as Alzheimer's
or Huntington's disease
F.U. Hartl & M. Hayer-Hartl (2002) Science 295, 5561
Molecular Chaperones in the Cytosol
Aggregation of nonnative protein chains as a side-reaction of productive folding in
the crowded environment of the cell.
F.U. Hartl & M. Hayer-Hartl (2002) Science 295, 5561
Aggregation of misfolded proteins
in neurodegenerative diseases
(A) Alzheimer's disease. Arrow, extracellular amyloid plaque. (B) Fibrillar tau inclusions in Pick's
disease. (C) PrPSc amyloid deposition in prion disease. (D) Multiple Lewy bodies in a nigral
neuron in Parkinson's disease. (E, F) Neuronal intranuclear inclusions of mutant ataxin-3 in
Machado-Joseph's disease.
JP Taylor et al. (2002) Science 296, 1991
Medicine: Danger — misfolding proteins
Protein folding is vital to living organisms. But errors in this process
generate misfolded structures that can be lethal.
R.J. Ellis & T.J.T. Pinheiro (2002) Nature 416, 483
La enfermedad de Alzhemier
APP
Ab
b

Secretasas
byg
g
membrana
Agregación
anormal
Placas
seniles
Teoría baptista:
El corte aberrante de la proteína precursora del amiloide
(APP) mediante dos proteasas (las secretasas beta y gamma) da lugar al fragmento
beta-amiloide (Ab , cuya agregación origina la placa senil. La secretasa alfa corta
APP de modo normal.
J. Ávila, Diario de Sevilla, 11 Enero 2001
Alzheimer's and amyloid
Amyloid-b peptides (Ab) come in a variety of sizes, of which the 42amino-acid form (Ab42) is thought to contribute significantly to the
development of Alzheimer's disease.
B. Strooper & G. König (2002) Nature 414, 159
La enfermedad de Alzhemier
Quinasas
Tau
Agregación
Tau
fosforilada
Ovillos
neurofibrilares
Teoría taoísta: La proteína Tau ayuda a mantener el armazón estructural de las
neuronas. Cuando se fosforila, la proteína Tau se agrega y aparecen los ovillos
neurofibrilares, haciendo que las neuronas cambien de forma y dejen de funcionar.
J. Ávila, Diario de Sevilla, 11 Enero 2001
Alzheimer's and amyloid
Strategies for reducing the levels
of amyloid peptides in the body.
All have been validated in animal
models.
B. Strooper & G. König (2002) Nature 414, 159
Amyloid diseases
Many human disorders — a well-known example being Alzheimer's
disease — are characterized by the misfolding and aggregation of
key proteins
Structure of the pentamer
of the serum amyloid P
protein (SAP)
L. Iversen (2002) Nature 417, 231
Targeted pharmacological depletion of serum amyloid
P component for treatment of human amyloidosis
This palindromic compound
(CPHPC) crosslinks and
dimerizes SAP molecules.
The normal plasma protein serum amyloid P component (SAP)
binds to fibrils in all types of amyloid deposits, and contributes
to the pathogenesis of amyloidosis.
M.B. Pepys et al. (2002) Nature 417, 254
Targeted pharmacological depletion of serum amyloid
P component for treatment of human amyloidosis
Two SAP pentamers crosslinked by five molecules of CPHPC
M.B. Pepys et al. (2002) Nature 417, 254
Targeted pharmacological depletion of serum amyloid
P component for treatment of human amyloidosis
Whole-body 123I-labelled SAP scintigraphy. At 6 h, the blood pool
background is completely absent and the liver, which is the only site
of catabolism of SAP in vivo, has taken up the tracer.
M.B. Pepys et al. (2002) Nature 417, 254
Prión normal (izqda) y anómalo (drcha)
Possible routes
of the BSE
prion from
cows to the
human brain
Prion replication and spread
Prion replication and spread
Disulfide Bridge
(Cys179 – Cys214)
Normal Human Prion Protein (PrPC)
(PDB entry 1QLX)
Prion replication and spread
Cys179
Cys214
Oxidized Prion Protein
(PDB entry 1QLX)
Reduced Prion Protein
(PDB entry 1I4M)
Prion replication and spread
Dimerization of Human Prion Protein
The dimer results from the 3D swapping of the C-terminal helix 3 and
rearrangement of the disulfide bond
Prion replication and spread
Speculative model
for conversion of
PrPC to a PrPRDX
fibril
Lee & Eisenberg ( 2003) Nature Struct. Biol. 10, 725 - 730