Protein folding & unfolding

Protein folding is the physical process by which a protein chain is translated to its native three-
dimensional structure, typically a "folded" conformation by which the protein becomes
biologically functional.
Via an expeditious and reproducible process, a polypeptide folds into its characteristic three-
dimensional structure from a random coil. Each protein exists first as an unfolded polypeptide
or random coil after being translated from a sequence of mRNA to a linear chain of amino
acids.
At this stage the polypeptide lacks any stable (long-lasting) three-dimensional structure.
As the polypeptide chain is being synthesized by a ribosome, the linear chain begins to fold
into its three-dimensional structure.
Folding of many proteins begins even during translation of the polypeptide chain. Amino acids
interact with each other to produce a well-defined three-dimensional structure, the folded
protein, known as the native state.
The resulting three dimensional structure is determined by the amino acid sequence or
primary structure.
The correct three-dimensional structure is essential to function, although some parts of
functional proteins may remain unfolded, so that protein dynamics is important.
Failure to fold into native structure generally produces inactive proteins, but in some
instances misfolded proteins have modified or toxic functionality.
Several neurodegenerative and other diseases are believed to result from the accumulation of
amyloid fibrils formed by misfolded proteins.
Amyloid fibrils are insoluble protein aggregates, and their formation may cause a variety of
human degenerative disorders including Alzheimer's disease.
Many allergies are caused by incorrect folding of some proteins, because the immune system
does not produce antibodies for certain protein structures.
Denaturation of proteins is a process of transition from the folded to the unfolded state. It
happens in cooking, in burns, in proteinopathies , and in other contexts. The duration of the
folding process varies dramatically depending on the protein of interest.
When studied outside the cell, the slowest folding proteins require many minutes or hours to
fold primarily due to proline isomerization, and must pass through a number of intermediate
states, like checkpoints, before the process is complete. On the other hand, very small single-
domain proteins with lengths of up to a hundred amino acids typically fold in a single step.
Time scales of milliseconds are the normal and the very fastest known protein folding
reactions are complete within a few microseconds.

Process of protein folding

Primary structure
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.

Secondary structure
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 are 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.
αhelices are formed by hydrogen bonding of the backbone to form a spiral shape. The β
pleated sheet is a structure that forms with the backbone bending over itself to form the
hydrogen bonds.
The hydrogen bonds are between the amide hydrogen and carbonyl oxygen of the peptide
bond. There exists anti-parallel β pleated sheets and parallel β pleated sheets where the
stability of the hydrogen bonds is stronger in the antiparallel β sheet as it hydrogen bonds with
the ideal 180 degree angle compared to the slanted hydrogen bonds formed by parallel
sheets.
Tertiary structure
The alpha helices and beta pleated sheets can be amphipathic in nature, or contain a
hydrophilic portion and a hydrophobic portion.
This property of secondary structures aids in the tertiary structure of a protein in which the
folding occurs so that 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. 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 "co-assembly" of subunits that have already folded;
in other words, multiple polypeptide chains could interact to form a fully functional
quaternary protein.
Driving forces of protein folding
Folding is a spontaneous process that is mainly guided by hydrophobic interactions, formation
of intramolecular hydrogen bonds & van der Waals forces.
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 depends
upon the solvent , the concentration of salts, the pH, the temperature, the possible presence
of cofactors and of molecular chaperones.

Hydrophobic effect
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. 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).
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.
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.
Hydrophobic effect Play media Example of a small eukaryotic heat shock protein In proteins
with globular folds, hydrophobic amino acids tend to be interspersed along the primary
sequence, rather than randomly distributed or clustered together.
However, proteins that have recently been born de novo, which tend to be intrinsically
disordered, show the opposite pattern of hydrophobic amino acid clustering along the primary
sequence.

Chaperones
Molecular 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 catalyst proteins, which catalyze chemical
reactions responsible for slow steps in folding pathways.
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 of peptide group.
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 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.
Heat shock proteins have been found in all species examined, from bacteria to humans,
suggesting that they evolved very early and have an important function. 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.
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 (i.e. confinement), 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 Chaperones 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.

Protein misfolding and neurodegenerative disease

A protein is considered to be misfolded if it cannot achieve its normal native state. This can be
due to mutations in the amino acid sequence or a disruption of the normal folding process by
external factors. The misfolded protein typically contains β-sheets that are organized in a
supramolecular arrangement known as a cross-β structure.
These β-sheet-rich assemblies are very stable, very insoluble, and generally resistant to
proteolysis.
The structural stability of these fibrillar assemblies is caused by extensive interactions between
the protein monomers, formed by backbone hydrogen bonds between their β-strands.
The misfolding of proteins can trigger the further misfolding and accumulation of other
proteins into aggregates or oligomers.
The increased levels of aggregated proteins in the cell leads to formation of amyloid-like
structures which can cause degenerative disorders and cell death.
The amyloids are fibrillary structures that contain intermolecular hydrogen bonds which are
highly insoluble and made from converted protein aggregates.
Therefore, the proteasome pathway may not be efficient enough to degrade the misfolded
proteins prior to aggregation.
Misfolded proteins can interact with one another and form structured aggregates and gain
toxicity through intermolecular interactions.
Aggregated proteins are associated with prion-related illnesses such as Creutzfeldt–Jakob
disease, bovine spongiform encephalopathy (mad cow disease), amyloid-related illnesses such
as Alzheimer's disease and familial amyloid cardiomyopathy or polyneuropathy, as well as
intracellular aggregation diseases such as Huntington's and Parkinson's disease.
These age onset degenerative diseases are associated with the aggregation of misfolded
proteins into insoluble, extracellular aggregates and/or intracellular inclusions. It is not
completely clear whether the aggregates are the cause or merely a reflection of the loss of
protein homeostasis, the balance between synthesis, folding, aggregation and protein
turnover.