Previous page: Considerations on the kinetics of protein folding
Protein folding in vitro
Protein folding in vivo, the role of molecular chaperones
Protein folding in in vitro experiments can be different
from that of inside the cells. The methods, mainly used in folding experiments,
can be seen in one of the previous sections.
Following the in vitro folding can lead us to very important discoveries
in this field, but in the interpretation of the data we have to be very
careful, as the environment of the folding protein is very different under
in vitro and in vivo circumstances. The major difference is, that in in
vitro experiments there is only one protein, with an unfolded structure,
which cannot interact with other components of the solvent. On the other
hand, there are a lot of interactions in the cell between different proteins,
during the folding process. The viscosity of the cytoplasm is rather different
from that of the solvents, used in in vitro experiments, the molecular
crowding makes the cytoplasm just like "honey". The proteins in the experiments
are folded, or they can be denatured by chemical or physical methods, which
may not reflect the in vivo circumstances of folding totally. There are
some proteins that need a special help in the folding process, this help
is given by molecular chaperones.
Protein folding in vivo, the role of molecular chaperones
The chaperones are major prokaryotic and eukaryotic
proteins, with the function of helping in folding of nascent polypeptide
chains, helping refolding of denatured proteins, and preventing aggregation
of surface-exposed hydrophobic parts of proteins, having problems with
folding. Chaperones help the proteins to fold, so they increase the speed
of folding, by stabilizing unstable intermediates of the appropriate polypeptide
chain, and decreasing the activation-energy barriers during folding. They
do not change the thermodynamics of folding, ie. the ratio of folded and
unfolded polypeptides, they only influence the kinetics of gyration. In
this sense they are often correlated with the enzymes. However, sometimes
they are very similar to them, but sometimes are very different, as they
are not too specific for the ligands, they help to fold, the substrates
are very large, and their large-scale functions make them key-molecules
of the cells. Their unspecifity is very good, since a protein can fold
incorrectly in a lot of ways, so there may be a lot of incorrect intermediates,
but the correct folding can occur only in one way in most cases. Mostly
they recognize hydrophobic surfaces on the proteins, and prevent them from
aggregation.
Beside this function, chaperones can play an important
role in signal transduction, in the maintenance of the organized state
of the cytoplasm and other intracellular compartments, in the motions inside
the cell, and some other vital functions of the cells. Sometimes they are
called stress proteins, or heat shock proteins, because their synthesis
increases (in most of the cases) after various forms of cellular stress,
such as heat, cold, detergents, increase of ionic strength, changes in
pH, toxic agents. However, the termini are not equivalent to each other,
as some of the chaperones' level does not change upon stress, in these
some cases they are called heat shock cognate proteins (referring to the
state, that they are homologous to heat shock proteins, but their synthesis
does not depend on stress). The hsp abbreviation is used in the first,
and the hsc in the second case. The grp-s (glucose regulated proteins)
are chaperones, that function in the endoplasmic reticulum. They are more
or less the homologues of the cytoplasmic chaperones. There are cold shock
proteins as well, they are abbreviated as csp-s, their role is in some
kind different from that of the hsp-s. (As at lower temperature the stability
of hydrogen bonds increase according to the Boltzmann-function, the intra-
and intermolecular associations can get stronger, which is unfavourable,
when the dissociation of these molecules, such as different strands of
RNA is required. This problem is solved by the cold shock proteins.) Let
us read some things on the major families of chaperones in prokaryotic
and eukaryotic organisms.
These are the most important chaperone families:
Ubiquitin, (and the family of hsp8)
The ubiquitin is a 76-amino acid long polypeptide,
which plays important role in the regulation of intracellular degradation
of cytoplasmic proteins. It is called sometimes hsp8, as its relative molecular
weight is 8500 Da, but this terminus is not very common. The ubiquitin
functions coordinately with the proteasome, the 20S and 26S proteasomes.
In the first step the molecule is activated by ATP, then it makes crosslinks
through its lyzine residues with the proteins to be degraded. In front
of the proteasome, there is also a chaperone-complex, which recognises
the ubiquitinated proteins, and therefore coordinates them to the inner
cavity of the proteasome, where the degradation happens. The ubiquitin
can also activate the proteases, however, the regulation of this small
chaperone is understood only in a few cases. It plays role in the degradation
of cyclins, and in this way it has major function in the cell-cycle regulation
as well.
According to a recent article, it coordinates the
degradation of approximately 30% of newly synthesized polypeptides and
proteins, as this fraction makes serious mistakes during the first steps
of folding and needs to be degraded in order to maintain the cellular homeostasis.
The ubiquitin is a small, 8 kDa weight protein, with
an alpha-helix and beta-sheet in its structure.
GroES and the corresponding hsp10 family
This family is further described in the chapter on
GroEL
and hsp60, as they form a large structural and functional complex together.
Hsp25, hsp27, hsp32, the small molecular chaperones
These chaperones called small molecular chaperones,
as their molecular weight is in the range between 25 and 32 kDa. The hsp32
is an enzyme, the hem-oxygenase, it oxidizes the hem part of hemoglobin
into bilirubin, which is an antioxidant agent. It regulates the NADPH-concentration
in the cell, as well. Therefore it plays important role in the antioxidant
defense mechanisms of the cell.
The hsp25 and hsp27 are members of the hsp28 protein-family.
The crystallin is also the member of this family. They take part in the
regulation of apoptosis (Mehlen, Schulze-Osthoff & Arrigo, 1996)
and in the reorganization of the actin filaments. They are regulated by
a phosphorylation-dephosphorylation cycle, and by changes in gene expression.
The phosphorylated form tends to be monomeric, whilst the dephosphorylated
form is multimeric (Kato, Hasegawa, Goto & Inaguma, 1994). So
the phosphorylation upon extracellular signals leads to a dissociation
of the multimeric protein. The monomers can act then as chaperones.
The hem-oxigenase. The hem group is bound between alpha-helical
structures.
The DnaJ, and its eukaryotic counterpart, the hsp40
are strongly related to the function of the DnaK/hsp70 family and the major
cytoplasmic chaperone-complex, the foldosome.
So, their function is mentioned in that chapter.
The collagenin is the chaperon of the collagen macromolecule, and therefore is expressed in larger quantities in those cells, which synthesize collagen. The synthesis and the postsynthetic modification of collagen is rather complicated, which requires the help of chaperones. The hsp47 can be found in the endoplasmic reticulum of the cells in a phosphorylated form. It binds very strongly the type I. and IV. procollagen molecules, and possesses a serine-protease inhibitor activity. So it prevents the procollagen extension peptides from being degraded, which is essential in the later formation of quaternary structure of collagen molecules outside the cell. It dissociates from the procollagen only in the Golgi-vesicles.
The interesting structure of the hsp47, which is a
serine-proteinase inhibitor.
PPI-s, the peptidil-prolil-cis-trans-isomerases
The PPI-s are members of the large, cytosolic chaperone complex, the foldosome so they can be found there, as well as the:
PDI-s, the protein-disulfide-isomerases
The GroEL molecule is a 60 kDa moleculer weight protein,
which forms tetradecamers. In this tetradecameric structure there are two
toroids above each other. This structure has a point group 72 symmetry.
There is a channel inside the toroids, this is the place, which is responsible
for the chaperone's activity. The GroES-molecules form a heptamer, which
is bound to the GroEL-tetradecamer on one side of it, as a cap. The interaction
with the appropriate GroEL ring influences the symmetry as well, so the
GroEL-GroES complex has a single point group 7 symmetry. However,
there are forms, when both the two GroES-binding sites of the GroEL
molecule binds the GroES-heptamer, but the function of this complex is
not known (Török, Vígh & Goloubinoff, 1996).
The chaperone-activity of hsp60 is strongly coupled with an ATP-ADP-exchange
cycle, however there are some theories existing about the precise details
of this phenomenon. The protein to be chaperoned gets into the cavity of
the chaperone-complex, which is then isolated from the environment by the
contact with the GroES-heptamer. Upon the energy of the ATP, the molecule
makes "breathing motions", in this way it helps the incorrectly folded
polypeptide chain to unfold, which gives the possibility of a correct refolding
after this cycle. This "percolator"
model gives important functions to the water molecules inside and outside
the chaperonin as well (Csermely, 1999). The nucleotides, required
for the chaperone-cycle are exchanging through the small holes between
the subunits of the GroEL-GroES complex.
If you are interested on excellent motion pictures
of hsp60, browse, the site of the Birkbeck
College for GroEL, the motions of the molecule can be seen in a delightful
manner. If you are very interested in motion pictures of molecules, you
can browse the Database
of Macromolecular Movements of the Yale Gerstein Lab, although there
has not been created a coordinate file of a folding protein, yet.
The DnaK, and its eukaryotic counterpart, the hsp70,
are among the most important chaperones of the cell. The hsp70 can be found
mainly in the cytoplasm, but their are forms, that take place in intracellular
organelles, for example the endoplasmic reticulum or the mitochondria.
This form is called mthsp70, according to its location. Its major role
is different in bacteria and in eukaryotes. As the bacteria's ribosomes
does not have chapeone-activity, the folding of nascent polypeptide chains
is fully required the help of the DnaK and other chaperones in the plasma
of the prokaryotic organism (Deuerling, Schulze-Speching, Tomoyasu,
Mogk & Bukau, 1999). As the eukaryotic ribosome possesses a chaperone-activity,
the function of the cytosolic hsp70 is to help in the refolding of the
incorrectly folded nascent or older polypeptide chains. In bacteria, therefore
the mutation of hsp70 is often lethal (Deuerling et al, 1999). There
are some other chaperones, making contacts with the hsp70, such as the
hsp40 (in prokaryotes the DnaJ), which helps the hsp70 in recognition of
the substrates. These are the introductive steps in the formation of the
large citoplasmic chaperone complex, the foldosome. Other components are
the Hip and Hop proteins in the first steps, which gives the possibility
of interaction with the cytoplasm's next important chaperone, the hsp90.
The p23 also joins the complex, which then is able to bind cytoplasmic
proteins, such as the steroid receptor, and a lot of signalling molecules,
among others kinases, phosphatases and transcription factors. The complex
can associate with PPI-s (peptidil-prolil-cis-trans-isomerases), such as
the FKBP51, FKBP52 and Cyp40 (cyclosporin A-binding protein). These are
small chaperones, which function is to catalize the isomerisation of the
cis-trans peptide bonds adjacent to proline residues. As mentioned in correlation
with the proline, the cis-configuration
of peptide bonds in case of proline residues leads in an extraordinary
turn of the polypeptide chain, which can be good in case of reverse turn
structures, but in other cases it can hinder the protein folding. This
activation free-energy barrier is very high in normal conditions, so it
could lead to considerable folding traps during folding. The PPI-s give
the solution to this problem, as they can "catalize the geometrical isomerisation
of the amide bonds. This leads to an acceleration in protein folding.
The protein-disulfide-isomerases (PDI) accelerate formation
of the correct pattern of disulfide bonds
in the protein, which can be very complicated sometimes. They occur both
in citoplasm and in the endoplasmic reticulum, as mainly the extracellular
proteins have disulfide bonds in the oxidative environment.
The hsp70's function is coupled to an ATP-ase cycle
as well as that of the hsp60's. Its two domains are very different in respect
to their secondary structures. The synthesis of hsp70 is increased after
stress. Its homologue in the endoplasmic reticulum is the grp78, which
helps in the transport and folding of extracellular proteins.
The DnaK has two domains, one of which consisiting
of alpha-helices, the other consisting of beta-sheets. The beta-sheet containing
domain has a peptide-binding capability. You can see the aligned packing
of beta-sheets on the picture.
The hsp90 is a very abundant citosolic chaperone, 2-3% of the eukaryotic citoplasm is made of this protein. Nevertheles, it can be transported to the nucleus, where it might act as a nuclear chaperone as well (Schnaider et al, 1999). There is an interesting discussion about its ATP-binding and ATP-ase ability. It is known for years, that its N-terminal domain can bind ATP (Csermely & Kahn, 1991; Prodromou et al, 1997), but recently a new ATP-binding site in the C-terminus has also been discovered. It interacts with a large scale of substrates, such as other chaperones and signalling molecules, the compounds of cytoskeleton and microfilamentous structures. It makes interactions with histones (Schnaider et al, 1999), and according its DNA-binding capability is also investigated (personal communication of T. Schnaider). It can bind peptides as well. Its major role is to take part in the composition of the foldosome, and to prevent aggregation of partially unfolded proteins by making interactions with them. The loss-of-function mutation of hsp90 is lethal in the early embrionic age, which sheds light on its fundamental, although not yet completely known function. Just as the hsp70, it also has a homologue in the endoplasmic reticulum (grp94), which is important in the folding of extracellular proteins, mainly glycoproteins.
The hsp90 has three distinct domains, the N-terminal domain's structure was resolved in 1997. The figure shows the N-terminal domain. The helices are indicated by red. There is a cleft between helices and the eight-stranded beta-sheet. This cleft is capable of bindind ATP and ADP. The ADP molecule is shown by a spacefill model in the figure.
If you are interested more on this chaperone, you can find some more,
interesting details on this site.
The Clp-families, the hsp100 and 110
The hsp100 or hsp110 is an approximately 104 kDa
molecular weight protein, which forms hexamers. It maintains the cell's
thermotolerance. Not only does it protect from the aggregation, but it
often desaggregates the aggregated structures. For this activity it needs
the energy of ATP.
Its function is often told to be similar to that of the hsp60's. In
the prokaryotes, its homologues are the Clp-proteins (ClpA, ClpB, ClpX,
ClpP). It can form complexes with other chaperones, such as hsp40 or hsp70
(Glover
& Lindquist, 1998).