PROTEIN
FOLDING
Proteins become functional only when they assume a distinct tertiary
structure. Many physiologically and therapeutically important proteins present
their surface for recognition by interacting with molecules such as
substrates, receptors, signaling proteins and cell–surface adhesion
macromolecules. When recombinant proteins are produced in Escherichia coli,
they often form inclusion bodies into which they are deposited as insoluble
proteins. Formation of such insoluble states does not naturally occur in cells
where they are normally synthesized and transported. Therefore, an in vitro
process is required to refold insoluble recombinant proteins into their native,
physiologically active state. This is usually accom-plished by solubilizing the
insoluble proteins with detergents or denaturants, followed by the
purifica-tion and removal of these reagents concurrent with refolding the
proteins.
Unfolded states of proteins are usually highly stable and soluble in the
presence of denaturing agents. Once the proteins are folded correctly, they are
also relatively stable. During the transition from the unfolded form to the
native state, the protein must go through a multitude of other transition
states in which it is not fully folded and denaturants or solubilizing agents
are at low concentrations or even absent.
The refolding of proteins can be achieved in various ways. The dilution
of proteins at high denaturant concentration into aqueous buffer will decrease
both denaturant and protein concentration simultaneously. The addition of an
aqueous buffer to a protein–denaturant solution also causes a decrease in
concentrations of both denaturant and protein. The difference in these
procedures is that, in the first case, both denaturant and protein
concentrations are the lowest at the beginning of dilution and gradually
increase as the process continues. In the second case, both denaturant and
protein concentrations are high-est at the beginning of dilution and gradually
decrease as the dilution proceeds. Dialysis or the diafiltration of protein in
the denaturant against an aqueous buffer resembles the second case, since the
denaturant concentration decreases as the procedure continues. In this case,
however, the protein concen-tration remains unchanged. Refolding can also be
achieved by first binding the protein in denaturants to a solid phase, i.e., to
a column matrix, and then equilibrating it with an aqueous buffer. In this case,protein
concentrations are not well defined. Each procedure has advantages and
disadvantages and may be applicable for one protein, but not to another.
If proteins in the native state have disulfide bonds, cysteines must be
correctly oxidized. Such oxidation may be done in various ways, e.g., air
oxidation, glutathione catalyzed disulfide exchange, or adduct formation
followed by reduction and oxidation or by disulfide reshuffling.
Protein folding has been a topic of intensive research since Anfinsen’s
demonstration that ribonu-clease can be refolded from the fully reduced and
denatured state in in vitro experiments. This folding can be achieved only if
the amino acid sequence itself contains all information necessary for folding
into the native structure. This is the case, at least partially, for many
proteins. However, a lot of other proteins do not refold in a simple one-step
process. Rather, they refold via various intermediates which are relatively
com-pact and possess varying degrees of secondary structures, but which lack a
rigid tertiary structure. Intrachain interactions of these preformed secondary
structures eventually lead to the native state. However, the absence of a rigid
structure in these preformed secondary structures can also expose a cluster of
hydrophobic groups to those of other polypeptide chains, rather than to their
own poly-peptide segments, resulting in intermolecular aggre-gation. High
efficiency in the recovery of native protein depends to a large extent on how
this aggregation of intermediate forms is minimized. The use of chaperones or
polyethylene glycol has been found quite effective for this purpose. The former
are proteins, which aid in the proper folding of other proteins by stabilizing
intermediates in the folding process and the latter serves to solvate the
protein during folding and diminishes interchain aggregation events.
Protein folding is often facilitated by co-solvents, such as
polyethylene glycol. As described above, proteins are functional and highly
hydrated in aqueous solutions. True physiological solutions, how-ever, contain
not only water but also various ions and low and high molecular weight solutes,
often at very high concentrations. These ions and other solutes play a critical
role in maintaining the functional structure of the proteins. When isolated
from their natural environment, the protein molecules may lose these
stabilizing factors and hence must be stabilized by certain compounds, often at
high concentrations. These solutes are also used in vitro to assist in protein
folding, and to help stabilize proteins during large-scale purification and
production as well as for long-term storage. Such solutes are often called
co-solvents when used at high concentrations, since at such high concentrations
they also serve as a solvent along withwater molecules. These solutes encompass
sugars, amino acids, inorganic and organic salts, and polyols. They may not
strongly bind to proteins, but instead typically interact weakly with the
protein surface to provide significant stabilizing energy without inter-fering
with their functional structure.
When recombinant proteins are expressed in eukaryotic cells and secreted
into media, the proteins are generally folded into the native conformation. If
the proteins have sites for N-linked or O-linked glycosylation, they undergo
varying degrees of glycosylation depending on the host cells used and level of
expression. For many glycoproteins, glycosy-lation is not essential for
folding, since they can be refolded into the native conformation without
carbo-hydrates, nor is glycosylation often necessary for receptor binding and
hence biological activity. However, glycosylation can alter important
biological and physicochemical properties of proteins, such as
pharmacokinetics, solubility, and stability.
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