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-helices and 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 cellCytosolic proteinsIn 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 proteinsProteins 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 byWhat 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 -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.