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Protein folding in the cell
The field of protein folding has changed dramatically
over the past 25:
1. Since the late 1980s, it has become clear, for example,
that many proteins require assistance to fold in the
cell and that this is provided by helper proteins
(chaperones).
This is not to say that Anfinsen’s original observation that
folding is a spontaneous event is erroneous but that the
normally spontaneous folding process needs assistance in
the cell.
Why is folding in vivo so difficult?
1. First, folding and biosynthesis are intimately coupled and the newly emerging polypeptide
chains can fold before synthesis is complete. As soon as enough amino acids protrude from
the ribosome, they will fold to the lowest available energy minimum for that length of chain.
For short sequences, this might involve secondary structure formation but, for longer
sequences, the lack of complete sequence information will inevitably lead to incorrect
tertiary structure formation. In some cases, an entire domain could fold co-translationally.
For a multidomain protein, however, the absence of a partner domain could expose a
dangerous hydrophobic surface, especially in the highly concentrated environment of the
polysome.
2. A second danger is caused by the highly crowded environment of the cellular milieu. At these
concentrations, the thermodynamic activity (effective concentration) of partially folded
states is increased 10–100-fold. As a consequence, the intermolecular binding constants
between partially folded states are increased, leading to an increased probability of
aggregation during folding.
The saviours of folding in the cell are the molecular chaperones. These proteins assist the
folding process in vivo by taking part in the biosynthetic process and by capturing misfolded
states in the cytosol post-translationally and allowing them renewed opportunities to fold.
They are also important in protecting proteins from aggregation as a consequence of heat
stress and play a role in a host of important cellular events, including signal transduction,
targeting and protein degradation
Folding Accessory proteins
1. Protein disulfide isomerase. Has a thioredoxin domain
(2 cys residues) proximal to a hydrophobic patch, presumably for
binding random coil conformations of unfolded protein.
2. Prolyl cis-trans isomerase. (also PPIase) is an enzyme found in both
prokaryotes and eukaryotes that interconverts the cis and trans isomers of
peptide bonds with the amino acid proline. Proteins with prolyl isomerase
activity include cyclophilin, FKBPs, and parvulin
3. Chaperones. Over 20 types of molecular chaperones.
Prevents improper association of surface exposed hydrophobic
patches.
Domain boundaries of PDI aligned
for human (top) and yeast (bottom)
PDI. Domains a (orange) and a’
(yellow) are homologous to TRX
and contain the catalytic CxxC
motif (green). Domains b (dark
blue) and b’ (light blue) also adopt
a TRX fold, but do not share high
sequence similarity with each other
or with domains a or a’. The flexible
linker region x (black) is located
between domains b’ and a’. The
link between domains a and b is
only one residue. The C-terminal
extension (red) contains a (K/H)DEL
retention signal for the ER. (b)
Ribbon diagram based on the
crystal structure of yeast PDI
showing the active-site cysteines in
green space-filling representation.
Colors of the domains are the same
as in (a).
The bacterial machinery for oxidative protein folding. Oxidized DsbA (light blue with yellow active site, 1FVK) catalyzes the
formation of disulfide bonds in newly secreted proteins by transferring its active-site disulfide. The resulting reduced DsbA
(light blue with red active site, 1A2L) is reoxidized by the inner membrane protein DsbB (green), which then passes its
electrons on to quinones. Terminal cytochrome oxidases reoxidize reduced quinones and transfer electrons to molecular
oxygen. In proteins with more than one pair of cysteines, DsbA can introduce incorrect disulfide bonds, thereby trapping
proteins in non-native conformations. The isomerase DsbC corrects non-native disulfide bonds by catalyzing disulfide
shuffling (dark blue with yellow active site, oxidized DsbC, PDB 1EEJ; dark blue with red active site, reduced DsbC, PDB
1TJD). DsbC is maintained in an active reduced form by the inner membrane protein DsbD (consisting of a, b and g
domains). This protein uses NADPH as a source of reducing power: electrons flow successively from NADPH via TRX (gray
with red active site, PDB 2TIR) to the DsbD domains b (black), g (grayblue with red active site, PDB 1L6P) and then a (grayblue with red active site, PDB 1L6P).
The eukaryotic machinery for oxidative protein folding in the ER. Oxidized PDI (slate blue with yellow active
site; yeast, PDB 2B5E) acts as a disulfide donor in the oxidative folding (oxidation) of reduced proteins.
Reduced PDI (slate blue with red active site) can shuffle disulfide bonds (isomerization). PDI is reoxidized
by a cascade that consists of Ero1 (mammals) or Ero1p (green; yeast, PDB 1RP4), its cofactor flavin adenine
dinucleotide (FAD) and molecular oxygen. Electrons flow from the CxxC catalytic motif of PDI to the CxxxxC
motif (red, reduced; yellow, oxidized) of Ero1, which is located in a flexible loop, and from there to a ‘rigid’
CxxC motif in the interior of Ero1. Finally, electrons flow from the rigid CxxC motif to FAD, and oxygen serves
as the terminal electron acceptor. In yeast, the active-site disulfide bond on the flexible loop of Ero1p can
engage in disulfide exchange with the cysteines in the active site of PDI in a so called ‘disulfide shuttle
mechanism’. The Ero1p–PDI interaction is thought to be facilitated by domain a’ of PDI. The resulting mixed
disulfide between Ero1p and PDI can resolve into reduced Ero1p and reoxidized PDI via an intramolecular
thiol–disulfide exchange reaction. After regeneration, PDI is ready to enter another cycle of substrate
disulfide oxidation.
Interestingly, isolated a and a’ domains can reduce
and oxidize protein disulfide bonds, but they
cannot shuffle disulfides. At least to some extent,
domain b’ in combination with domains a’ and c is
needed for isomerase activity, although this
combination of domains is not sufficient for
complex isomerase reactions involving large
conformational changes .
There are some organism-specific differences
among PDIs that relate mainly to the way in which
the catalytic domains contribute to enzyme
catalytic activity. For example, the redox properties
of domains a and a’ are similar to each other in
mammalian PDI but differ in yeast PDI.
In addition to its redox properties, PDI acts as a
chaperone. Its chaperone activity does not require
the catalytic cysteines of either domain. For
human PDI, it has been suggested that all
domains contribute to its chaperone role, but the
C-terminal end of the protein, including domains a’
and c, are important.
Schematic diagram of de novo folding in the
cytosol of eukaryotes and prokaryotes.
(a) Folding that does not depend on hsp70 or
hsp60. (b) Folding assisted by hsp70 or trigger
factor (TF). (c) Folding assisted by both hsp70 or
TF and an hsp60 chaperonin.
The diagram shows common features between
chaperone-assisted folding in prokaryotes and
eukaryotes, although there are differences. For
example, TF is found in prokaryotes whereas
nascent-chain-associated protein (NAC) and
GimC are found in eukaryotes.
In both prokaryotes and eukaryotes, a significant
proportion of proteins fold without the assistance of
either hsp70 or the chaperonins (a). In other
proteins, folding of the polypeptide chain might
involve proteins of the hsp70 class and/or TF (b).
In addition, interaction with hsp60 chaperonins is
required to fold complex proteins (c). Known
substrates of hsp60 include actin and tubulin in
eukaryotes, and an array of proteins involved in
transcription, translation and metabolic enzymes in
prokaryotes. Co-translational domain folding and the
association of the nascent chain with chaperonins is
favoured in eukaryotes.
GroEL is a 14-mer of identical
58 kDa subunits arranged as
two stacked rings. The whole
assembly is held together by
the back-to-back rings of
equatorial domains that form
most of the intra-ring and all
of the inter-ring contacts
The structure of an extended polypeptide binding to the hydrophobic patch on the apical
domain of GroEL.
(a) The molecular surface of the GroEL apical domain, showing hydrophobic binding sites
on helices H and Ι, is coloured according to surface curvature. Convex, concave and
flat regions are shown in green, grey and white, respectively. Bulky side chains on the
bound peptide insert into hydrophobic cavities on the GroEL apical domain
(b) The location of helices H and Ι on a ribbon diagram of the rear three subunits of the
GroEL–GroES–ADP crystal structure
Crystal structures of Escherichia coli GroEL (Hsp60)
and its complex with ADP and GroES (Hsp10).
(a) The three domains of the GroEL subunit are colour
coded – green for the equatorial domain, yellow for the
intermediate domain and red for the apical domain. The
equatorial domain contains the nucleotide-binding site,
shown occupied, as well as the two inter-ring contacts
at the base of the molecule (negatively charged
residues in red, positive in blue). The apical domain
contains the hydrophobic binding sites (grey, spacefilling representation) for non-native polypeptides. ATP
is shown in pink. (b) The structure of the unbound
GroEL 14-mer, with three subunits shown at the front.
The hydrophobic binding sites face into the end
cavities. (c) The GroEL–ADP–GroES complex , with
one GroEL subunit and one GroES subunit (cyan).
Range of conformational changes in
the GroEL subunit, showing the
(a) unbound GroEL
(b) GroEL–ATP
(c) GroEL–ADP–GroES .
The domains move as rigid bodies
about the two hinge points marked
with arrows in (a). Hydrophobic
substrate-binding residues are
shown in grey and nucleotides in
pink. In the ATP-bound state (b), the
binding sites twists towards the
viewer but, in the GroES-bound
state (c), the apical domain is
twisted in the opposite direction so
that the binding sites face away
from the direction of view. In (b), the
hinge residues are omitted because
the local conformation is unknown.
Allosteric structural changes caused by
ATP binding to GroEL. (a,b) Unbound
GroEL and GroEL (D398A)–ATP. Cryoelectron-microscopy density maps are
shown as transparent blue surfaces and
the domains of three subunits, docked
into the maps as rigid bodies, are colour
coded – green for the equatorial domain,
yellow for the intermediate domain and
red for the apical domain. Notice the
contact between adjacent intermediate
and apical domains in the top ring of (a),
and the switch to a contact with the
adjacent equatorial domain in (b) (black
circles). The ATP-bound complex is
asymmetric and extended vertically. The
inter-ring interface is also distorted. (c,d)
Top views of the same structures. ATP
binding causes a large anticlockwise twist
of the apical domains. The hydrophobic
residues (circled) become less accessible
in the ATP-bound state (d).
Animazione
GroEL ATPase and the folding cycle. The GroE complexes are shown as sections through the
stacked-ring complexes.
(a) GroEL has high affinity for non-native polypeptide substrate (black curved line).
(b) ATP binds with positive co-operativity to one ring but negative co-operativity between
rings, producing an altered conformation with reduced substrate affinity.
(c) The ATP-bound ring rapidly binds GroES, simultaneously sequestering the hydrophobic
binding sites and encapsulating the substrate in the folding chamber. There is a massive
conformational change in the GroES-bound ring.
(d) The substrate folds inside the chamber and ATP is hydrolysed.
(e) ATP binding to the opposite ring primes the release of GroES and the trapped substrate.
(f) A new substrate gets encapsulated
Potential for modulation of the hydrophobic effect inside chaperonins.
Biophys J. 2008 Oct;95(7):3391-9.
England JL, Pande VS.
Despite the spontaneity of some in vitro protein-folding reactions, native folding in vivo
often requires the participation of barrel-shaped multimeric complexes known as
chaperonins. Although it has long been known that chaperonin substrates fold upon
sequestration inside the chaperonin barrel, the precise mechanism by which confinement
within this space facilitates folding remains unknown. We examine the possibility that the
chaperonin mediates a favorable reorganization of the solvent for the folding reaction. We
discuss the effect of electrostatic charge on solvent-mediated hydrophobic forces in an
aqueous environment. Based on these physical arguments, we construct a simple,
phenomenological theory for the thermodynamics of density and hydrogen-bond order
fluctuations in liquid water. Within the framework of this model, we investigate the effect
of confinement inside a chaperonin-like cavity on the configurational free energy of water
by calculating solvent free energies for cavities corresponding to the different
conformational states in the ATP-driven catalytic cycle of the prokaryotic chaperonin GroEL.
Our findings suggest that one function of chaperonins may involve trapping unfolded
proteins and subsequently exposing them to a microenvironment in which the hydrophobic
effect, a crucial thermodynamic driving force for folding, is enhanced.
Conformational diversity and protein evolution – a 60-year-old hypothesis
revisited
Leo C. James 1 and Dan S. Tawfik 2
Trends in Biochemical Sciences 2003, 28:361-368
Complex organisms have evolved from a limited number of
primordial genes and proteins. However, the mechanisms by
which the earliest proteins evolved and then served as the origin
for the present diversity of protein function are unknown. Here,
we outline a hypothesis based on the 'new view' of proteins
whereby one sequence can adopt multiple structures and
functions. We suggest that such conformational diversity could
increase the functional diversity of a limited repertoire of
sequences and, thereby, facilitate the evolution of new proteins
and functions from old ones.
The ‘simplistic view’ of proteins assumes an energy diagram with a single well (a global minimum), which
corresponds to the existence of a single structural conformer (a).
The ‘new view’ of proteins has an energy landscape with many local minima corresponding to an ensemble
of pre-existing structures with similar but discrete energy levels (plasticity; b). The mechanism by which
conformational changes are linked to function also varies according to the two views.
The induced-fit model has become part of the ‘simplistic view’ because it assumes that, in the absence of a
ligand (L), the protein (P) adopts one conformation only. The active conformer (P*) is induced by ligand
binding and has no existence in the absence of the ligand. By contrast, the ‘new view’, assumes that both P
and P* are pre-existing conformers (preequilibrium) and that ligand binding shifts the equilibrium only in
favour of P*.
Proposed model of enzyme evolution mediated by conformation diversity and functional
promiscuity.
(a) The enzyme is in equilibrium between different conformations. The native substrate (yellow)
selects the dominant conformer (dark blue) and, thus, enzyme activity confers selective advantage.
(b) An alternative conformation potentiates the binding of a second substrate (pink). The secondary
activity confers a limited selective advantage under changing environmental conditions.
(c) Gene duplication enables one copy to evolve improved activity with the promiscuous substrate
while the original gene maintains its original function.
The ‘new view’ of proteins has prompted the revision of many facets of protein science. Here,
we have outlined the intriguing implications that this ‘new view’ might have for protein
evolution. The hypotheses described are supported by many properties of today’s proteins,
including the recent demonstration of a linkage between conformational diversity and multispecificity in antibodies
Conformational diversity of antibody SPE7. Two different conformations of SPE7 allow it to bind
to entirely unrelated antigens from across the structural spectrum (hapten to protein). One
conformation (a) has a deep and narrow binding site that binds the hapten against which SPE7
was raised [dinitrophenol (DNP)]. The other conformation (b) has a flat binding surface that
promiscuously binds a protein antigen (grey). The difference in the binding surfaces can be seen
at the centre of each image; there is a deep cavity in the DNP-bound structure that is absent in
the protein-bound structure.
Intrinsically unstructured proteins
Peter Tompa
Trends in Biochemical Sciences, 2002, 27:527-533
The recent suggestion that the classical structure–function paradigm should be
extended to proteins and protein domains whose native and functional state is
intrinsically unstructured has received a great deal of support. There is ample
evidence that the unstructured state, common to all living organisms, is
essential for basic cellular functions; thus it deserves to be recognized as a
separate functional and structural category within the protein kingdom. In this
review, recent findings are surveyed to illustrate that this novel but rapidly
advancing field has reached a point where these proteins can be comprehensively
classified on the basis of structure and function.
Intrinsically unstructured, or pliable, proteins (IUP) are distinct
from folded proteins with a well-defined three-dimensional
structure. Their commonness and clear structural, functional and
evolutionary separation justify their recognition as a unique
protein category
These proteins are characterized by an almost complete lack of
folded structure and an extended conformation with high
intramolecular flexibility and little secondary structure.
Unstructured proteins/regions have no enzymatic activity as the proper
spatial organization of active site residues requires a rigid fold they
cannot provide. Furthermore, their functions are invariably linked to
their structural disorder and can be classified into 28 distinct categories.
Here, it is suggested that they actually fall into five broad functional
classes based on their mode of action
1. The first class is that of entropic chains, with functions that directly stem from
disorder and thus fall outside the realm of folded proteins.
2. The IUPs in the other four categories function via molecular recognition; as noted,
the target/partner with which they bind with high specificity can be DNA, RNA,
another protein, or a range of small ligands. On binding, IUPs can alter the action of
their partner in a variety of ways.
1. Some of them, the effectors, modify the activity of a single partner protein or
assembled proteins; so far, only inhibitors appear to belong to this second
class.
2. The third class is that of scavengers, which store and/or neutralize small
ligands.
3. The fourth class are the assemblers, which assemble, stabilize and regulate
large multiprotein complexes such as the ribosome, cytoskeleton, transcription
preinitiation complex, chromatin, and even the extracellular matrix.
The benefits of being unstructured
Structural disorder is essential for IUPs as their various functions stem either
directly from this state or from some local folding/ordering in molecular
recognition.
1. The uncontested advantage of the lack of a folded structure is realized in
entropic chain functions, which depend directly on the disordered state and
are thus out of reach of globular proteins.
2. Often, globular domains are connected by flexible linkers/spacers in
multidomain proteins; these regions regulate distance and enable
unprecedented freedom in orientational search.
3. Another unique functional faculty of IUPs is that their open structure is
largely preserved when they complex with their target, which provides for a
disproportionately large binding surface and multiple contact points for a
protein of the given size
4. An additional prominent feature is that IUPs can adopt different structures
upon different stimuli or with different partners, which enables their
versatile interaction with various targets
Intrinsically unstructured proteins (IUPs) contact their target over
a large surface area
p27Kip1 (in yellow) complexed with cyclin-dependent kinase 2
(Cdk2) and cyclin A