SUPPLEMENTS (Full Edition)
4 Protein folding
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- A cofactor is a small inorganic component (often a metal ion) that is required for the proper structure or function of an enzyme.
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Chaperones are a class of proteins
which bind to incompletely folded or assembled proteins in order to
assist their folding or prevent them from aggregating.
- A domain of a protein is a discrete continuous part of the amino acid sequence that can be equated with a particular function.
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We can consider two principles that might control the folding of a protein into the correct higher-order structure.
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Folding is an intrinsic feature of the primary sequence.
In this case, the final structure must always be the most stable
thermodynamically and can be generated at any time after synthesis of
the polypeptide chain is complete.
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The correct structure can be generated only during the synthesis of the polypeptide.
Then it becomes possible that an intrinsically less stable structure
could prevail because the protein becomes "trapped" in it during
synthesis.
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The relationship between higher-order structures and the primary structure may be revealed
when a protein is denatured
by heating or by chemical treatments that disrupt protein conformation.
Most denaturing events involve the breakage of hydrogen and other
noncovalent bonds. An exception is the disruption of S – S bridges that
results from treatment with reducing agents. However, all of these
changes affect the conformation; the primary sequence of amino
acids in the polypeptide chain remains unaltered.
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In some cases, the higher-order structure
follows ineluctably from the primary sequence. The enzyme ribonuclease
is the classic example (1115). After the protein has been denatured,
its active conformation can be regained by reversing the denaturing procedure.
All the information necessary to form the secondary structure resides in the primary sequence.
Thus the production of active ribonuclease is an inevitable event
whenever the intact primary chain is placed in the appropriate
conditions.
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In other cases, proteins can be
irreversibly denatured. Thus under certain (nonphysiological)
conditions, a protein may have alternative stable conformations. In
some cases the correct conformation probably can be attained only
during synthesis of the protein. The conformation could depend on
specific interactions between regions of the protein that can occur
only in the absence of other regions (that is, those that have not yet
been synthesized). This is probably the more common situation.
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In some instances, a cofactor
that is part of the active protein (such as the iron-binding heme group
of the cytochromes) must be present in order for the polypeptide chain
to take up its proper conformation. In the case of multimeric proteins,
it may be necessary for one subunit to be present in order for another
to acquire the proper conformation.
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Protein folding is usually rapid in vivo, occurring within seconds or less.
It begins even before a protein has been completely synthesized. Probably it involves a
sequential folding
mechanism, in which the reaction passes through discrete (although
highly transient) intermediates. The process is initiated by the
collapse of hydrophobic side chains into the "core" of the protein;
this occurs within milliseconds. Units of secondary structure, largely
a-helices and ß-sheets,
form on the same time scale. The transition from this structure to the
final tertiary structure is slower. The process appears to be
cooperative, so that formation of one region of secondary structure
enhances formation of the next region, and so on (for review see 2389)
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Figure S10
Both catalytic and stoichiometric functions are required to assist protein folding
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The acquisition of structure when a
protein is synthesized is not a spontaneous process, but may require
assistance. More precisely, we should say that spontaneous folding is a
slow reaction, which under normal cellular conditions is a
rate-limiting step. The rate is significantly increased by several
types of additional functions. These are summarized in Figure S10.
They fall into two groups: enzymes that catalyze specific isomerization
steps; and factors that act stoichiometrically to influence folding
directly.
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Figure S11
Formation of a disulfide bridge between the sulfhydryl
groups of two cysteines may connect different parts of a polypeptide
chain.
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The formation of disulfide bonds is shown in Figure S11.
The animation shows that formation of a disulfide bond may have a major
effect on the conformation of the protein. It is influenced by both
environment and specific accessory proteins. Disulfide bonds are rare
in cytoplasmic proteins, but common in exported proteins. This is
related to a difference in the thiol/disulfide redox state between
internal and external conditions. It may help to prevent bond formation
in a bacterium, but helps to drive it in the periplasm (the layer
surrounding the bacterial cell).
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Disulfide bond formation can occur spontaneously in vitro,
but the rate is slow. It has a t
>15 min, compared with the ability to form disulfide bonds correctly within a few seconds
in vivo. The process is catalyzed in vivo
by an enzyme, protein disulfide isomerase (PDI). This is a curious
protein, which participates in a variety of functions concerned with
protein-modification, in addition to its sponsorship of disulfide
bridge formation. It is not entirely clear whether it simply helps the
initial formation of disulfide bonds or whether it also catalyzes
rearrangement of disulfide bonds that have formed incorrectly (for
review see 3443)
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Figure S12
The configuration of proline has an important effect on protein conformation.
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Proline has a major effect upon protein
structure because of the restrictions imposed by its ring structure.
Proline introduces a bend in a polypeptide chain, because the nitrogen
atom is restrained by the ring structure. The existence (and
interconversion) of two stereochemical forms of the peptidyl-proline
link is an important feature of protein structure. The direction of the
bend is determined by whether the proline is in the cis or trans
configuration, as shown in Figure S12.
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Proteins containing proline fold slowly
because the peptidyl-proline link does not necessarily form in correct
stereochemical conformation. The enzyme peptidyl-prolyl isomerase (PPI)
catalyzes the cis-trans conversion, and by this
means significantly accelerates the folding reaction. Enzymes with PPI
activity fall into two major groups, named for their abilities to bind
certain drugs: cyclophilin PPI binds the drug cyclosporin A, and FKBP
PPI binds the drug FK506. Members of the cyclophilin class are better
characterized, and they vary in their specificity of action from those
that appear to be generic (able to act on any protein) to those that
appear to work only with specific proteins. This makes the point that,
although control of proline isomerization is a general feature of many
proteins, it can also be used to control specifically the maturation of
an individual protein.
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Proteins that act stoichiometrically on the
folding of other proteins are called molecular chaperones.
A chaperone forms a complex with a protein during folding,
but is required only during assembly, and is not part of the mature structure.
The major role of a chaperone is to prevent the formation of
incorrectly folded structures, in which the substrate protein might
otherwise become trapped during folding (see Figure 8.8 in
Chaperones may be required for protein folding).
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Protein folding is an intricate process.
The primary sequence of a protein is a crucial determinant of its
higher-order structures. Sometimes it is the sole determinant, but in
most cases additional interactions are involved in acquiring the final
conformation. In each case, however, if the primary sequence is
synthesized within the appropriate environment, it will acquire the
proper higher-order structures.
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Although higher-order structure follows
from primary sequence, the same general tertiary structure can be
determined by different primary sequences. For example, the globin (red
blood cell) proteins of different species vary substantially in
sequence, but have the same general tertiary structure.
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An important concept is that a protein may consist of domains.
A domain is a (relatively) independent region of the protein. In some
cases its conformation can be acquired independently by the relevant
fragment of the polypeptide chain. Some globular proteins consist of
discrete domains connected by "clefts." Sometimes a substrate binds to
the cleft between domains.
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Figure S13
Overlapping arrangements of discrete domains are found in proteins.
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A domain may represent a functional unit
that is identified with a particular activity of the protein, for
example, its ability to perform a certain catalytic activity, to bind a
certain ligand, or to interact specifically with other types of
domains. The lengths of recognized domains vary from 30 – 300 amino
acid residues. Certain types of domains may be found in proteins with
particular locations; for example, on the exterior of the cell. A
domain may represent an evolutionary unit. It may have arisen as a
functional polypeptide or region of a polypeptide and later have
associated with other domains to generate a new protein with additional
abilities. The occurrence of closely related domains in different
proteins is common; Figure S13 compares the use of domains in two blood cell proteins.
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2389 Fersht, A. R. and Daggett, V.
(2002).
Protein folding and unfolding at atomic resolution.
Cell 108, 573-582.
PubMed Journal
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3443 Sevier, C. S. and Kaiser, C. A.
(2002).
Formation and transfer of disulphide bonds in living cells.
Nat. Rev. Mol. Cell Biol. 3, 836-847.
PubMed Journal
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1115 Anfinsen, C. B.
(1973).
Principles that govern the folding of protein chains.
Science 181, 223-230.
PubMed
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© Jones and Bartlett Publishers (2007)
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