Download Folding in the cell Cytosolic proteins

How do proteins fold?
Folding in a test-tube
The structure of proteins is determined by the amino acid sequence;
many proteins in solution can be unfolded by heat and other
denaturants such as high concentrations of urea and guanidinium
chloride, but they will spontaneously refold on returning conditions
to normal. This refolding takes place in two phases. First a very rapid
formation of secondary structure such as a-helices and b-sheets,
and folding of these to form a compact shape, which is presumed to
be driven by hydrophobic collapse, so that most of the interior of the
protein is occupied by hydrophobic amino acids, which have a low
affinity for water. The second phase is slower; the secondary structure
elements interact with each other to form the native tertiary structure
Folding in the cell
Cytosolic proteins
In the cell proteins are synthesized on ribosomes. Free ribosomes make proteins
that stay in the cytosol, and secreted and membrane proteins are made by
ribosomes that become attached to the membrane of the endoplasmic reticulum
(ER). Small single domain cytosolic proteins do not usually refold in vitro if they
are missing the C-terminus, so probably do not begin to fold before synthesis is
complete; but in larger multidomain proteins the first domains are often folded
before the last part of the protein has been made. The initial fast folding of the
polypeptide is followed by slower steps, the formation of disulphide bonds between
cysteines, and the change of some of the proline residues to the cis form. In the cell
these steps are catalysed by the enzymes protein disulphide isomerase (PDI) and
peptidyl proline isomerase (PPI). In vivo, the concentration of protein at the site
of synthesis is very high, and as hydrophobic residues exposed in the unfolded
protein are likely to interact, there is competition between correct folding
and aggregation. This is seen when cloned genes are expressed at high levels in
E.coli; the normal machinery is overwhelmed, and protein precipitates out as
inclusion bodies. Aggregation also occurs in some diseases.
Secreted and membrane proteins
Proteins that are destined for export from the cell, such as secreted
proteins, or those that are to be translocated to locations such as
membranes, or other cellular compartments, have to stay unfolded
until they have crossed the ER membrane (in the case of secreted
proteins) or the organelle membrane. These proteins have a signal
sequence at the N-terminus, which docks with a signal recognition
particle (SRP) that then stops elongation until the complex
(ribosome-mRNA-nascent chain-SRP) has reached a receptor for
SRP on the ER membrane. The SRP is displaced when the
ribosome complex is bound to the membrane and translation
continues, the polypeptide being extruded into the lumen of the ER,
the signal sequence is removed and the protein can then fold.
In the case of membrane proteins the protein remains in the
membrane (a stop transfer sequence stops translocation,
and the protein remains anchored in the membrane). In
some cases the C terminus remains in the cytosol, in
others a loop from the middle of the protein is inserted,
leaving both N and C ends in the cytosol. Proteins with
multiple membrane spanning regions have a succession of
signal and stop transfer sequences (apart from the first one,
the signal sequences do not get cleaved off the protein).
Misfolding of proteins can be caused by
What can go wrong?
•Mutation in the DNA so that the amino acid sequence differs
from normal.
•Lack of an enzyme or chaperone needed to fold a protein.
•Correctly folded protein becoming misfolded by accumulated
damage due to oxidation or other chemical reaction.
•Interaction with other misfolded protein.
•
Misfolding can cause disease in several ways:
•A protein is non-functional
•A protein is in short supply.
•A protein is unable to get to the right place, due to inability to
fold correctly.
•The presence of protein aggregates, that have a damaging
effect on the cell.
•One important factor to keep in mind:
Defective and misfolded proteins are normally rapidly
degraded by proteolysis. It may therefore not always be
obvious that a protein is misfolding, it may look as if it is
absent.
Inherited Diseases
Usually a total absence of a protein will be lethal, so large deletions in the DNA will
usually alter the protein so drastically that the embryo does not survive. In general a
small change, such as the mutation of one amino acid or a small deletion will not
inactivate a protein unless it is in the active site of an enzyme, a ligand binding site or
in an essential structural position (such as a sharp turn where only certain
conformations can occur, or in amino acids involved in salt bridges in the interior of
the molecule). Many single amino acid mutations have little affect on the folding, but
in a number of cases it has been shown that a mutation produces a defect in folding,
that results in a less efficient, or unstable protein. In some others the effect is to cause
an abnormal interaction, so that a new type of structure is formed.
Inherited diseases have examples of all four types of consequences of misfolded protein:
•Emphysema is due to a lack of a-anti-proteinase.
•Osteogenesis imperfecta is due to an unstable fold.
•Cystic fibrosis, and familial hypercholesteremia may be due to a misfolded protein
that cannot take up its proper position.
•Familial amyloidosis, Creuzfelt-Jacob disease, and Alzheimer's disease involve
aggregation of misfolded proteins.