Protein folding is the process by which a polypeptide chain folds into its characteristic and functional three-dimensional structure. It involves four stages: primary structure is the amino acid sequence; secondary structure involves folding into alpha helices and beta sheets through hydrogen bonding. Tertiary structure involves folding of secondary structures into the final three-dimensional structure. Some proteins undergo quaternary structure formation through interactions between folded polypeptide chains. The folding process is driven by the hydrophobic effect and other non-covalent interactions and allows proteins to attain biologically functional conformations. Chaperone proteins assist in protein folding to facilitate efficient folding in living cells.

2.  Protein folding is a process in which a
polypeptide folds into a specific, stable,
functional, three-dimensional structure.
 It is the process by which a protein structure
assumes its functional shape or
conformation.

3.  Proteins are much more complicated than just a chain of amino-
acids because proteins fold spontaneously depending on the R
groups in their amino-acid sequence. The structure of proteins is
very important for their function. The protein folding process can
be divided up into four stages, which we can think of as stages in
putting together a book:
 Primary structure: a chain of amino-acids joined together (letters
coming together to form words)
 Secondary structure: the chains fold into sheets or coils
(sentences)
 Tertiary structure: the sheets or coils fold in on one another
(chapters of a book) 4. Quaternary structure: amino-acid chains
folded in their tertiary structure interact with one another to give
the final functional protein (e.g. haemoglobin has 4 chains) (a
book)

4.  The primary structure of a protein, its linear amino-acid
sequence, determines its native conformation. The specific
amino acid residues and their position in the polypeptide
chain are the determining factors for which portions of the
protein fold closely together and form its three
dimensional conformation.
 The amino acid composition is not as important as the
sequence. The essential fact of folding, however, remains
that the amino acid sequence of each protein contains the
information that specifies both the native structure and
the pathway to attain that state.
 This is not to say that nearly identical amino acid
sequences always fold similarly. Conformations differ
based on environmental factors as well; similar proteins
fold differently based on where they are found.

5.  Formation of a secondary structure is the first step in the
folding process that a protein takes to assume its native
structure.
 Characteristic of secondary structure is the structures known
as alpha helices and beta sheets that fold rapidly because
they are stabilized by intramolecular hydrogen bonds, as was
first characterized by Linus Pauling.
 Formation of intramolecular hydrogen bonds provides
another important contribution to protein stability. Alpha
helices are formed by hydrogen bonding of the backbone to
form a spiral shape (refer to figure on the right).
 The beta pleated sheet is a structure that forms with the
backbone bending over itself to form the hydrogen bonds (as
displayed in the figure to the left). The hydrogen bonds are
between the amide hydrogen and carbonyl carbon of the
peptide bond.

6.  They exists anti-parallel beta pleated sheets and parallel
beta pleated sheets where the stability of the hydrogen
bonds is stronger in the anti-parallel beta sheet as it
hydrogen bonds with the ideal 180 degree angle
compared to the slanted hydrogen bonds formed by
parallel sheets.
 The alpha helices and beta pleated sheets can be
amphipathic in nature, or contain a hydrophilic portion
and a hydrophobic portion.
 This property of these secondary structures aids in the
folding of the protein as it aligns the helices and sheets in
such a way where the hydrophilic sides are facing the
aqueous environment surrounding the protein and the
hydrophobic sides are facing the hydrophobic core of the
protein. Secondary structure hierarchically gives way to
tertiary structure formation.

7.  Once the protein's tertiary structure is formed and
stabilized by the hydrophobic interactions, there may
also be covalent bonding in the form of disulfide
bridges formed between two cysteine residues.
 Tertiary structure of a protein involves a single
polypeptide chain; however, additional interactions of
folded polypeptide chains give rise to quaternary
structure formation.
 Quaternary structure
 Tertiary structure may give way to the formation of
quaternary structure in some proteins, which usually
involves the "assembly" or "coassembly" of subunits
that have already folded; in other words, multiple
polypeptide chains could interact to form a fully
functional quaternary protein.

8.  Folding is a spontaneous process independent of energy inputs
from nucleoside triphosphates. The passage of the folded state
is mainly guided by hydrophobic interactions, formation of
intramolecular hydrogen bonds, van der Waals forces, and it is
opposed by conformational entropy.
 The process of folding often begins co-translationally, so that
the N-terminus of the protein begins to fold while the C-
terminal portion of the protein is still being synthesized by the
ribosome; however, a protein molecule may fold spontaneously
during or after biosynthesis.
 While these macromolecules may be regarded as "folding
themselves", the process also depends on the solvent (water or
lipid bilayer), the concentration of salts, the pH, the temperature,
the possible presence of cofactors and of molecular chaperones.
 Proteins will have limitations on their folding abilities by the
restricted bending angles or conformations that are possible.
These allowable angles of protein folding are described with a
two-dimensional plot known as the Ramachandran plot, depicted
with psi and phi angles of allowable rotation

9.  Protein folding must be thermodynamically favorable within a cell in
order for it to be a spontaneous reaction. Since it is known that
protein folding is a spontaneous reaction, then it must assume a
negative Gibbs free energy value. Gibbs free energy in protein folding
is directly related to enthalpy and entropy. For a negative delta G to
arise and for protein folding to become thermodynamically favorable,
then either enthalpy, entropy, or both terms must be favorable.
 Entropy is decreased as the water molecules become more orderly
near the hydrophobic solute.
 Minimizing the number of hydrophobic side-chains exposed to water
is an important driving force behind the folding process. The
hydrophobic effect is the phenomenon in which the hydrophobic
chains of a protein collapse into the core of the protein (away from
the hydrophilic environment).
 In an aqueous environment, the water molecules tend to aggregate
around the hydrophobic regions or side chains of the protein,
creating water shells of ordered water molecules. An ordering of
water molecules around a hydrophobic region increases order in a
system and therefore contributes a negative change in entropy (less
entropy in the system).

10.  The water molecules are fixed in these water cages which drives
the hydrophobic collapse, or the inward folding of the
hydrophobic groups.
 The hydrophobic collapse introduces entropy back to the system
via the breaking of the water cages which frees the ordered
water molecules.
 The multitude of hydrophobic groups interacting within the core
of the globular folded protein contributes a significant amount to
protein stability after folding, because of the vastly accumulated
van der Waals forces (specifically London Dispersion forces).
 The hydrophobic effect exists as a driving force in
thermodynamics only if there is the presence of an aqueous
medium with an amphiphilic molecule containing a large
hydrophobic region. The strength of hydrogen bonds depends
on their environment; thus, H-bonds enveloped in a hydrophobic
core contribute more than H-bonds exposed to the aqueous
environment to the stability of the native state.

11.  Chaperones are a class of proteins that aid in the correct folding of other
proteins in vivo. Chaperones exist in all cellular compartments and interact with
the polypeptide chain in order to allow the native three-dimensional
conformation of the protein to form; however, chaperones themselves are not
included in the final structure of the protein they are assisting in.
 Chaperones may assist in folding even when the nascent polypeptide is being
synthesized by the ribosome. Molecular chaperones operate by binding to
stabilize an otherwise unstable structure of a protein in its folding pathway, but
chaperones do not contain the necessary information to know the correct native
structure of the protein they are aiding; rather, chaperones work by preventing
incorrect folding conformations.
 In this way, chaperones do not actually increase the rate of individual steps
involved in the folding pathway toward the native structure; instead, they work
by reducing possible unwanted aggregations of the polypeptide chain that might
otherwise slow down the search for the proper intermediate and they provide a
more efficient pathway for the polypeptide chain to assume the correct
conformations.
 Chaperones are not to be confused with folding catalysts, which actually do
catalyze the otherwise slow steps in the folding pathway. Examples of folding
catalysts are protein disulfide isomerases and peptidyl-prolyl isomerases that
may be involved in formation of disulfide bonds or interconversion between cis
and trans stereoisomers, respectively.

12.  Chaperones are shown to be critical in the process of protein folding in vivo
because they provide the protein with the aid needed to assume its proper
alignments and conformations efficiently enough to become "biologically
relevant". This means that the polypeptide chain could theoretically fold into
its native structure without the aid of chaperones, as demonstrated by protein
folding experiments conducted in vitro; however, this process proves to be
too inefficient or too slow to exist in biological systems; therefore,
chaperones are necessary for protein folding in vivo.
 Along with its role in aiding native structure formation, chaperones are shown
to be involved in various roles such as protein transport, degradation, and
even allow denatured proteins exposed to certain external denaturant factors
an opportunity to refold into their correct native structures.
 A fully denatured protein lacks both tertiary and secondary structure, and
exists as a so-called random coil. Under certain conditions some proteins can
refold; however, in many cases, denaturation is irreversible. Cells sometimes
protect their proteins against the denaturing influence of heat with enzymes
known as heat shock proteins (a type of chaperone), which assist other
proteins both in folding and in remaining folded. Some proteins never fold in
cells at all except with the assistance of chaperones which either isolate
individual proteins so that their folding is not interrupted by interactions with
other proteins or help to unfold misfolded proteins, allowing them to refold
into the correct native structure.

13.  This function is crucial to prevent the risk of precipitation into insoluble
amorphous aggregates. The external factors involved in protein
denaturation or disruption of the native state include temperature,
external fields (electric, magnetic), molecular crowding, and even the
limitation of space, which can have a big influence on the folding of
proteins.
 High concentrations of solutes, extremes of pH, mechanical forces, and
the presence of chemical denaturants can contribute to protein
denaturation, as well. These individual factors are categorized together as
stresses.
 Chaperones are shown to exist in increasing concentrations during times
of cellular stress and help the proper folding of emerging proteins as well
as denatured or misfolded ones.
 Under some conditions proteins will not fold into their biochemically
functional forms. Temperatures above or below the range that cells tend
to live in will cause thermally unstable proteins to unfold or denature (this
is why boiling makes an egg white turn opaque).
 Protein thermal stability is far from constant, however; for example,
hyperthermophilic bacteria have been found that grow at temperatures as
high as 122 °C, which of course requires that their full complement of vital
proteins and protein assemblies be stable at that temperature or above.

14.  Protein aggregation is a biological
phenomenon in which misfolded proteins
aggregate (i.e., accumulate and clump
together) either intra- or extracellularly.
 These protein aggregates are often
correlated with diseases. In fact, protein
aggregates have been implicated in a wide
variety of disease known as amyloidoses,
including ALS, Alzheimer's, Parkinson's and
prion disease.

15.  After synthesis, proteins typically fold into a particular three-dimensional
conformation that is the most thermodynamically favorable: their native state.
 This folding process is driven by the hydrophobic effect: a tendency for
hydrophobic (water-fearing) portions of the protein to shield itself from the
hydrophilic (water-loving) environment of the cell by burying into the interior of
the protein. Thus, the exterior of a protein is typically hydrophilic, whereas the
interior is typically hydrophobic.
 Protein structures are stabilized by non-covalent interactions and disulfide bonds
between two cysteine residues. The non-covalent interactions include ionic
interactions and weak van der waals interactions. Ionic interactions form between
an anion and a cation and form salt bridges that help stabilize the protein. Van der
waals interactions include nonpolar interactions (i.e. London dispersion forces)
and polar interactions (i.e. hydrogen bonds, dipole-dipole bond). These play an
important role in a protein's secondary structure, such as forming an alpha helix
or a beta sheet, and tertiary structure. Interactions between amino acid residues in
a specific protein are very important in that protein's final structure.
 When there are changes in the non-covalent interactions, as may happen with a
change in the amino acid sequence, the protein is susceptible to misfolding or
unfolding. In these cases, if the cell does not assist the protein in re-folding, or
degrade the unfolded protein, the unfolded/misfolded protein may aggregate, in
which the exposed hydrophobic portions of the protein may interact with the
exposed hydrophobic patches of other proteins. There are three main types of
protein aggregates that may form: amorphous aggregates, oligomers, and amyloid
fibrils.

16.  Protein aggregation can occur due to a variety of causes. There are four classes that these
causes can be categorized into, which are detailed below.
 1. Mutations
 Mutations that occur in the DNA sequence may or may not affect the amino acid sequence of
the protein. When the sequence is affected, a different amino acid may change the interactions
between the side chains that affect the folding of the protein. This can lead to exposed
hydrophobic regions of the protein that aggregate with the same misfolded/unfolded protein
or a different protein.
 In addition to mutations in the affected proteins themselves, protein aggregation could also be
caused indirectly through mutations in proteins in regulatory pathways such as the refolding
pathway (molecular chaperones) or the ubiquitin-proteasome pathway (ubiquitin ligases).
Chaperones help with protein refolding by providing a safe environment for the protein to
fold. Ubiquitin ligases target proteins for degradation through ubiquitin modification.
 2. Problems with protein synthesis
 Protein aggregation can be caused by problems that occur during transcription or translation.
During transcription, DNA is copied into mRNA, forming a strand of pre-mRNA that undergoes
RNA processing to form mRNA. During translation, ribosomes and tRNA help translate the
mRNA sequence into an amino acid sequence. If problems arise during either step, making an
incorrect mRNA strand and/or an incorrect amino acid sequence, this can cause the protein to
misfold, leading to protein aggregation.

17.  Environmental stresses
Environmental stresses such as extreme temperatures and pH or oxidative stress can also
lead to protein aggregation. One such disease is cryoglobulinemia.
Extreme temperatures can weaken and destabilize the non-covalent interactions between
the amino acid residues. pH outside of the protein's pH range can change the protonation
state of the amino acids, which can increase or decrease the non-covalent interactions.
This can also lead to less stable interactions and result in protein unfolding.
Oxidative stress can be caused by radicals such as reactive oxygen species (ROS). These
unstable radicals can attack the amino acid residues, leading to oxidation of side chains
(e.g. aromatic side chains, methionine side chains) and/or cleavage of the polypeptide
bonds. This can affect the non-covalent interactions that hold the protein together
correctly, which can cause protein destabilization, and may cause the protein to unfold.
 Aging
Cells have mechanisms that can refold or degrade protein aggregates. However, as cells
age, these control mechanisms are weakened and the cell is less able to resolve the
aggregates.
The hypothesis that protein aggregation is a causative process in aging is testable now
since some models of delayed aging are in hand. If the development of protein aggregates
was an aging independent process, slowing down aging will show no effect on the rate of
proteotoxicity over time.
However, if aging is associated with decline in the activity of protective mechanisms against
proteotoxicity, the slow aging models would show reduced aggregation and proteotoxicity.
To address this problem several toxicity assays have been done in C. elegans.
These studies indicated that reducing the activity of insulin/IGF signaling (IIS), a prominent
aging regulatory pathway protects from neurodegeneration-linked toxic protein
aggregation. The validity of this approach has been tested and confirmed in mammals as
reducing the activity of the IGF-1 signaling pathway protected Alzheimer's model mice
from the behavioral and biochemical impairments associated with the disease.

18.  There are two main protein quality control systems in the cell that are responsible
for eliminating protein aggregates. Misfolded proteins can get refolded by the bi-
chaperone system or degraded by the ubiquitin proteasome system or autophagy.
 Refolding
 The bi-chaperone system utilizes the Hsp70 (DnaK-DnaJ-GrpE in E. coli and Ssa1-
Ydj1/Sis1-Sse1/Fe1 in yeast) and Hsp100 (ClpB in E. coli and Hsp104 in yeast)
chaperones for protein disaggregation and refolding.
 Hsp70 interacts with the protein aggregates and recruits Hsp100. Hsp70 stabilizes
an activated Hsp100. Hsp100 proteins have aromatic pore loops that are used for
threading activity to disentangle single polypeptides. This threading activity can
be initiated at the N-terminus, C-terminus or in the middle of the polypeptide.
The polypeptide gets translocated through Hsp100 in a series of steps, utilizing
an ATP at each step. The polypeptide unfolds and is then allowed to refold either
by itself or with the help of heat shock proteins.
 Degradation[edit]
 Misfolded proteins can be eliminated through the ubiquitin-proteasome system
(UPS). This consists of an E1-E2-E3 pathway that ubiquinates proteins to mark
them for degradation. In eukaryotes, the proteins get degraded by the 26S
proteasome. In mammalian cells, the E3 ligase, carboxy-terminal Hsp70
interacting protein (CHIP), targets Hsp70-bound proteins. In yeast, the E3 ligases
Doa10 and Hrd1 have similar functions on endoplasmic reticulum proteins.

19.  Proteolysis is the breakdown of proteins into
smaller polypeptides or amino acids.
 Uncatalysed, the hydrolysis of peptide bonds
is extremely slow, taking hundreds of years.
Proteolysis is typically catalysed by cellular
enzymes called proteases, but may also occur
by intra-molecular digestion.
 Low pH or high temperatures can also cause
proteolysis non-enzymatically.

20.  The levels of proteins within cells are
determined not only by rates of synthesis, but
also by rates of degradation
 In eukaryotic cells, two major pathways—the
ubiquitin-proteasome pathway and lysosomal
proteolysis—mediate protein degradation.
 https://youtu.be/CA9RSzU3KaA?si=ravF89gn
NnqgdZab

21.  The major pathway of selective protein degradation in
eukaryotic cells uses ubiquitin as a marker that
targets cytosolic and nuclear proteins for rapid
proteolysis.
 Ubiquitin is a 76-amino-acid polypeptide that is
highly conserved in all eukaryotes (yeasts, animals,
and plants). Proteins are marked for degradation by
the attachment of ubiquitin to the amino group of the
side chain of a lysine residue.
 Additional ubiquitin are then added to form a
multiubiquitin chain. Such polyubiquinated proteins
are recognized and degraded by a large, multisubunit
protease complex, called the proteasome.
 Ubiquitin is released in the process, so it can be
reused in another cycle. It is noteworthy that both the
attachment of ubiquitin and the degradation of
marked proteins require energy in the form of ATP.

22.  Proteins are marked for rapid degradation by the covalent attachment of
several molecules of ubiquitin. Ubiquitin is first activated by the enzyme
E1.
 Activated ubiquitin is then transferred to one of several different
ubiquitin-conjugating enzymes (E2). In most cases, the ubiquitin is then
transferred to a ubiquitin ligase (E3) and then to a specific target
protein. Multiple ubiquitins are then added, and the polyubiquinated
proteins are degraded by a protease complex (the proteasome).

23.  Since the attachment of ubiquitin marks proteins for rapid
degradation, the stability of many proteins is determined by
whether they become ubiquitinated. Ubiquitination is a multistep
process.
 First, ubiquitin is activated by being attached to the ubiquitin-
activating enzyme, E1. The ubiquitin is then transferred to a
second enzyme, called ubiquitin-conjugating enzyme (E2). The
final transfer of ubiquitin to the target protein is then mediated
by a third enzyme, called ubiquitin ligase or E3, which is
responsible for the selective recognition of appropriate substrate
proteins.
 In some cases, the ubiquitin is first transferred from E2 to E3 and
then to the target protein (see Figure).
 In other cases, the ubiquitin may be transferred directly from E2
to the target protein in a complex with E3. Most cells contain a
single E1, but have many E2s and multiple families of E3
enzymes.
 Different members of the E2 and E3 families recognize different
substrate proteins, and the specificity of these enzymes is what
selectively targets cellular proteins for degradation by the
ubiquitin-proteasome pathway.

24.  A number of proteins that control fundamental cellular processes, such as
gene expression and cell proliferation, are targets for regulated
ubiquitination and proteolysis.
 An interesting example of such controlled degradation is provided by
proteins (known as cyclins) that regulate progression through the division
cycle of eukaryotic cells.
 The entry of all eukaryotic cells into mitosis is controlled in part by cyclin
B, which is a regulatory subunit of a protein kinase called Cdc2 . The
association of cyclin B with Cdc2 is required for activation of the Cdc2
kinase, which initiates the events of mitosis (including chromosome
condensation and nuclear envelope breakdown) by phosphorylating
various cellular proteins.
 Cdc2 also activates a ubiquitin-mediated proteolysis system that degrades
cyclin B toward the end of mitosis. This degradation of cyclin B inactivates
Cdc2, allowing the cell to exit mitosis and progress to interphase of the
next cell cycle.
 The ubiquitination of cyclin B is a highly selective reaction, targeted by a
9-amino-acid cyclin B sequence called the destruction box. Mutations of
this sequence prevent cyclin B proteolysis and lead to the arrest of dividing
cells in mitosis, demonstrating the importance of regulated protein
degradation in controlling the fundamental process of cell division.

25. The progression of eukaryotic cells
through the division cycle is controlled
in part by the synthesis and
degradation of cyclin B, which is a
regulatory subunit of the Cdc2 protein
kinase. Synthesis of cyclin B during
interphase leads to the formation of an
active cyclin B–Cdc2 complex, which
induces entry into mitosis. Rapid
degradation of cyclin B then leads to
inactivation of the Cdc2 kinase,
allowing the cell to exit mitosis and
return to interphase of the next cell
cycle.

26.  The other major pathway of protein degradation in eukaryotic cells
involves the uptake of proteins by lysosomes. Lysosomes are
membrane-enclosed organelles that contain an array of digestive
enzymes, including several proteases (see Chapter 9). They have several
roles in cell metabolism, including the digestion of extracellular proteins
taken up by endocytosis as well as the gradual turnover of cytoplasmic
organelles and cytosolic proteins.
 The containment of proteases and other digestive enzymes within
lysosomes prevents uncontrolled degradation of the contents of the cell.
Therefore, in order to be degraded by lysosomal proteolysis, cellular
proteins must first be taken up by lysosomes. One pathway for this
uptake of cellular proteins, autophagy, involves the formation of vesicles
(autophagosomes) in which small areas of cytoplasm or cytoplasmic
organelles are enclosed in membranes derived from the endoplasmic
reticulum (Figure 7.41). These vesicles then fuse with lysosomes, and
the degradative lysosomal enzymes digest their contents. The uptake of
proteins into autophagosomes appears to be nonselective, so it results
in the eventual slow degradation of long-lived cytoplasmic proteins.

27. Lysosomes contain various digestive
enzymes, including proteases.
Lysosomes take up cellular proteins by
fusion with autophagosomes, which
are formed by the enclosure of areas of
cytoplasm or organelles (e.g., a
mitochondrion) in fragments of the
endoplasmic reticulum. This fusion
yields a phagolysosome, which digests
the contents of the autophagosome.

28.  However, not all protein uptake by lysosomes is nonselective. For
example, lysosomes are able to take up and degrade certain
cytosolic proteins in a selective manner as a response to cellular
starvation.
 The proteins degraded by lysosomal proteases under these
conditions contain amino acid sequences similar to the broad
consensus sequence Lys-Phe-Glu-Arg-Gln, which presumably
targets them to lysosomes.
 A member of the Hsp70 family of molecular chaperones is also
required for the lysosomal degradation of these proteins,
presumably acting to unfold the polypeptide chains during their
transport across the lysosomal membrane.
 The proteins susceptible to degradation by this pathway are
thought to be normally long-lived but dispensable proteins.
Under starvation conditions, these proteins are sacrificed to
provide amino acids and energy, allowing some basic metabolic
processes to continue.