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Protein Folding and

Misfolding
BIOCHEM-700

Protein structure
Primary structure – simply the sequence, in order from N to C termini, of the amino acid residues.

British biochemist Frederick Sanger solved the procedure for determining the 1° structure of proteins using insulin as the
first accomplished (51 amino acids) in 1953. He was awarded the Nobel Prize for Chemistry in 1958 for this work and a
second Nobel Prize for Chemistry in 1980 for working out a similar protocol on sequencing nucleic acids, the only
person to receive two Nobel Prizes in the same field. Way to go, Fred!

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• Most proteins are made from 50-2,000 amino acids residues

• Residue: The part of amino acid left after engaging in peptide bond n
• Oligopeptide: Small number of amino acids linked together (from 5 to
20-50 amino acid residues)
• Polypeptide or protein: Longer amino acid chains (50 amino acid
residues and longer)
• Largest natural protein known is titin, 27,000 amino acids
• Functions as a scaffold for muscle proteins

Primary structure – amino acid sequence.

Secondary structure – backbone arrangements.

Tertiary structure – 3D shape.

Quaternary structure – multiple subunits.

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Primary structure of proteins

• The Primary Structure of Proteins • Peptide bonds are stable but require an
consist of amino acids linked by a input of energy to be formed
peptide bond (amide bond) • Protein’s polypeptide chain has
• The primary structure of proteins is directionality
the linear polymeric sequence of • The amino (-NH3 + ) terminal end is the
amino acid residues formed by beginning of the polypeptide chain
linking COO- (carboxyl) group of • The carboxyl (-COO- ) terminal end is the
one amino acids to the NH3 + end of the polypeptide chain
(amino) group of the adjacent
amino acids by a peptide bond. • The primary structure is always written
from the amino terminal to the carboxyl
• The formation of each peptide bond terminal or left to right
is accompanied by the loss of a
water molecule is called as
condensation reaction.

• Amino acid sequences have direction

• The pentapeptide Tyr-Gly-Gly-Phe-Leu shows the sequence from the
amino terminus to the carboxyl terminus
• It has 5 amino acid residues and 4 peptide bonds

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Molecular Weight Calculation of proteins and

poly peptides:
• The mean molecular weight for an amino acid is 110 g mol-1
• the molecular weight of a polypeptide can thus be estimated from the
number of its
(amino acid residues × 110 = ? g mol-1)
• The molecular weight of a polypeptide can also be measured in units of
Dalton (Da)
1 Da = 1 g mol-1 = 1 hydrogen atom
• Thus, protein with a molecular weight of 50,000 g mol-1 has a mass of 50,000
Da or 50 kDa
• Example:
=120 amino acids X 110
= 13,200 g mol-1 or Da
= 13.2 kDa

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Secondary structure of proteins

• Secondary structure is the two-dimensional structure stabilized by H-

bonds between amide protons (N—H) and carbonyl oxygens (C=O) of the
peptide bonds linking amino acid residues that are near one another in the
primary structure.
• Hydrogen bonds keep amino acids close to each other.
• Coiling or bending of the polypeptide chain leading to a specific structure.
Polypeptide chains can fold into regular structures.
• Example: Fibrous proteins are composed of an entirely secondary structure
pattern.
• Prominent examples of secondary structure:
• The α–helix
• The β-sheets
• Turns, loops and bends

α- Helix
• A rod-like structure formed by a tightly coiled
polypeptide chain arranged in a spiral
structure.
• Coil of the polypeptide chain in αhelical
structure brings 3.6 amino acids per turn
• It may be right-handed or left-handed.
• The alpha helix is a coiled structure stabilized
by intrachain H-bonds (H-bonds between
different parts of the same polypeptide chain)
• The α–helix is a tightly coiled rod-like
structure, with the R groups pointing out from
the axis of the helix
• Essentially all α–helices found in proteins are
right-handed (clockwise)
• All the backbone amide protons (N—H) and
carbonyl oxygens (C=O) of the peptide bond
form H-bonds except those at the end of the
helix
• The H-bonds are parallel to the axis of the
polypeptide helix

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Amino acids NOT found in α-helix

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β- pleated sheet
• β -pleated sheet may be composed of two or more peptide chains
• The β-pleated sheet can be parallel or antiparallel. Arrows Represent
from N to C terminal.
• In the parallel form; the chains run in the same direction.
• In the antiparallel one; the chains run in opposite directions

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• Optimal hydrogen bonding occurs when the sheet is bent (pleated) to

form a β-pleated sheet
• Beta sheets are stabilized by hydrogen-bonding (H-bonds) between
polypeptide strands (interchain = between chains not same chain)
• β-sheet is another common form of secondary structure
• β–sheets are formed by β–strands that are next to each other
• β-strands lying next to one another form a sheet ---Linked by H-bonds
between the carbonyl’s oxygen on one strand (C=O) and the amide’s
proton on the other strand (N—H)
• In contrast to an α-helix, the polypeptide in a β-strand is fully extended.
• β sheets can form between many strands, typically 4 or 5 but as many as
10 or more. The strands of a β-sheet may be parallel, antiparallel, or
mixed.

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Effect of amino acids

• Valine, threonine, and Isoleucine
with branched R groups at β-
carbon and the large aromatic
amino acids (phenylalanine,
tryptophan, and tyrosine) tend
to be present in β-sheets.
• Proline tends to disrupt β
strands
How are they illustrated/drawn?

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Antiparallel β sheets
• Adjacent β strands run in opposite directions
• H-bonds (green dashes) between NH and CO
groups, stabilize the structure
• In antiparallel sheets, H-bonds alternate
between being close together and wide apart
and are oriented approximately perpendicular
to the polypeptide backbone.

Parallel β sheets
• Adjacent β strands run in the same direction
• H-bonds (green dashes) connect amino acids on
different strands
• In a parallel sheet, The hydrogen bonds are
evenly spaced but slant in alternate directions.

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Mixed β-Sheets

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Tertiary Protein structure

Tertiary structure – overall three-dimensional shape (3D) of the
protein, mostly due to interactions between R-groups. Reveal information about the
biological functions of proteins and
“The spatial arrangement of amino acid residues that are their evolutionary origins
far apart in the sequence”
It is the result of interactions between the R-groups of the amino acid
residues in the peptide chain.
There are two main forms of tertiary structure; fibrous and globular
types Six types of interactions within the same chain are involved,
in order of
decreasing relative strength:
1. Disulfide bonds (R – S – S – R)
2. Electrostatics (salt bridges) between acidic
and basic R-groups
3. Hydrogen bonds between polar R-groups
4. Hydrophobic interactions between nonpolar Folds in such a fashion that
R-groups (most important for 3D structures.) distinct amino acids in its
5. Ionic bonding primary structure will come
6. Van der Waal forces into proximity.

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Quaternary structure of proteins

• Formation of subunits.
• “Quaternary structure a protein refers to the spatial arrangement of its
subunits”
• Quaternary structure – non-covalent organization/interaction among multiple
peptide chains. If only one peptide chain is present, there is no quaternary
structure.
• Proteins possess quaternary structure if they consist of 2 or more polypeptide
chains attached by non-covalent bonds, e.g. hemoglobin (4 subunits)
• Many proteins are composed of multiple polypeptide chains called subunits
• Such proteins are said to display quaternary structure
• Quaternary structure can be made up of two identical polypeptide chains
(homodimers) or different polypeptide chains (heterodimers)
• Quaternary structure refers to the arrangement of subunits and the nature of
their interactions

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Quaternary structure
Three types of interactions involved, in
order of decreasing relative strength:
1. Disulfide bonds (R – S – S – R)
2. Electrostatics (salt bridges) between
acidic and basic R-groups
3. Hydrogen bonds between polar R-
groups
4. Hydrophobic interactions between
nonpolar R-groups Ribbon drawing of carboxyltransferase from S. aureus.
(Purple) the α-chain; (gold) the β-chain.
B. K. Benson, G. Meades, Jr, A. Grove, and G. L. Waldrop
Why are multi-subunit proteins more common? Protein Sci. 2008 January; 17(1): 34–42
In large assemblies of proteins, such as collagen fibrils, it is easy
to construct subunits into huge protein
•Defects can be repaired by simply replacing the flawed Subunits either may function independently of each
subunit other or may work cooperatively, as in hemoglobin,
•Increasing the size of an enzyme through the -association of in which the binding of oxygen to one subunit of the
identical subunits is more efficient than increasing the length of tetramer increases the affinity of the other subunits
its polypeptide chain Important for enzyme regulation for oxygen

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Factors that determine protein structure

• The least amount of energy needed to stabilize the protein. This is
determined by:
• The amino acid sequence (the primary structure), mainly the internal
residues.
• 50% of the primary amino acid sequence is necessary just to define the 3-dimensional structure of a
typical protein.
• The classic in vitro studies of Anfinsen, who received a Nobel prize in 1972 in chemistry, demonstrated
that the primary amino acid sequence of protein contains all information necessary to direct the folding of
a polypeptide chain to the correct final structure
• The proper angles between the amino acids
• The different sets of weak non-covalent bonds that form between the
mainly R groups.
• Non-protein molecules.

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Protein folding
• Protein function is determined by how the
protein folds to form a specific three-
dimensional structure.
• The way that a protein folds is determined
by the free energy of the constituent amino
acid residues.
• Proteins are folded and held together by
several forms of molecular interactions.
Protein synthesis is a relatively
• The molecular interactions include slow process, proceeding at the
• the thermodynamic stability of the complex rate of 4 amino acids per second
• the hydrophobic interactions while the hydrophobic collapse of
• the disulfide bonds formed in the proteins. a protein can happen in
nanoseconds.

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Protein folding depends upon

• The solvent (water or lipid bilayer)
• The concentration of salt
• The PH
• The temperature
• The possible presence of a cofactor
• Molecular chaperones

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Driving force of protein folding

• Folding is a spontaneous process that is mainly guided
by hydrophobic interactions, the 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.

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Folding Catalysts accelerate the Rate of Folding

• Protein-folding catalysts (enzymes) that accelerate the rate-limiting steps in protein folding and avoid aggregation of
folding intermediates and Non-productive interactions with other proteins. These enzymes also assist cellular proteins to
avoid aggregation by accelerating the rate of correct folding.
• The two best-studied protein-folding catalysts are protein disulfide isomerase (EC5.3.4.1) and peptidyl-prolyl cis/trans-
isomerase (EC5.2.1.8).

Protein Disulfide Isomerases Peptidyl Prolyl Cis-Trans Isomerases (PPIs)

• Protein disulfide isomerase (PDI) is a member of the • PPIs are variously referred to as immunophilins such as cyclophilins and
thioredoxin superfamily of proteins and contains two FKBPs, rotamase, and parvulins.
copies of the characteristic active-site motif CXXC. • The term immunophilin comes from the ability of PPI to bind
• PDI facilitates folding through its ability to reduce or immunosuppressive drugs.
oxidize disulfide bridges in the presence of an • PPI participates in protein folding by accelerating the cis-trans
isomerization of prolyl peptide bonds.
oxidizing or reducing agent such as glutathione.
• Prolyl-isomerization is accelerated 300-fold by PPI. After isomerization,
• In eukaryotic cells, PDI is localized exclusively
the correct form of Prolyl is stabilized by the polypeptide secondary or
within the lumen of the rough ER. tertiary structure.
• Most PPI proteins are localized in the cytoplasm/nucleus, there are unique
forms present within the rough ER/nucleus, the plastid stroma, and the
mitochondrial matrix.

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Protein Folding in The Cytoplasm

• The majority of nuclear-encoded proteins reside in the cytoplasm throughout their lifetime.
• It is important to identify which molecular chaperones and folding catalysts assist the newly synthesized
cytoplasmic proteins to their final native state.
• Small, single domain and monomeric proteins can fold spontaneously in the cytoplasm without any
external assistance, or perhaps only with the assistance of a folding catalyst.
• Large proteins are recognized by the Stress70 chaperone machine and bound while still nascent.

Protein Folding in other Subcellular Compartments

• Most organellar proteins are nuclear-encoded and synthesized as precursors in the cytoplasm. As such,
they will interact with various cytoplasmic chaperones before membrane translocation.
• After membrane translocation, protein folding within other subcellular organelles of a plant cell likely
follows similar pathways as in the cytoplasm or ER, and Specific forms of HSP70, HSP90, the
chaperonins, and the small HSPs have been found in plastids, mitochondria, and peroxisomes.
• Proteins that fail to be properly folded are ultimately targeted for destruction.

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Role of Endoplasmic reticulum

• Protein secretion is defined by how plant cells help to build cell walls, store proteins in seeds and populate
organelles with proteins in the endomembrane system.
• The endoplasmic reticulum (ER) is the entry point for the secretory pathways and a compartment in which
proteins are introduced into ER by co-translational synthesis are folded.
• The conditions of temperature and pH, the salt concentration, and especially the total protein concentration found in vivo
(Plant) tend to promote a plethora of side reactions that compete with a single pathway that will lead to the correct final
structure.
• Protein folding in the ER is an error-prone, stochastic process (a highly efficient protein quality control system)
that operates to differentiate misfolded proteins from properly folded proteins.
• If several rounds of chaperone-assisted folding are ineffective, misfolded proteins are recognized and targeted
for degradation by proteasomes through the multi-step process called ER-associated protein degradation
(ERAD).
• Unfolded and partially folded proteins tend to aggregate when present at concentrations as high as 340 mg mL -1 in
Escherichia coli.

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Problem solvers: chaperones

• A Specialized group of proteins that are required for the proper folding of proteins.
• Many proteins (>100 amino acids) require the assistance of molecular chaperones in folding into their native
conformation and to avoid inappropriate protein-protein interactions.
• These proteins bind to polypeptide chains and help them fold with the most energetically favorable folding pathway.
• The molten globule state is an intermediate stage where a protein is “partly unfolded,” and it is thought that proteins are
in the molten globule state when recognized by chaperones or for membrane translocation.
• Chaperones also prevent the hydrophobic regions in newly synthesized protein chains from associating with each other
to form protein aggregates.
• Molecular chaperones, proteins that prevent inappropriate aggregation of exposed hydrophobic surfaces of unfolded or
partially folded proteins.
• Molecular Chaperons direct them into productive folding, transport, or degradation pathways, function to minimize
protein aggregation, and can promote the dissociation of aggregates that have formed.
Correctly
folded
protein
Polypeptide
Cap: GroES
Smaller peptide (<100 A.A) folds
by itself without any help.
Hollow
cylinder
GroEL
If refolding is not possible,
Molecular chaperons lead to Chaperonin
(fully assembled)
Steps of Chaperonin
Action:
2 The cap attaches, causing
the cylinder to change
3 The cap comes
off, and the
degradation of the protein. 1 An unfolded poly-
peptide enters the
shape in such a way that
it creates a hydrophilic
properly folded
protein is
cylinder from environment for the released.
one end. folding of the polypeptide.

Many diseases are the result of defects in protein folding

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Molecular Chaperones Prevent Non-productive Interactions

• Molecular chaperones only assist in the assembly/disassembly of proteins but are not themselves
components of the final structures.
• Molecular chaperones do not have an active role in protein folding, do not accelerate the folding
reactions, nor do they provide steric information directing protein folding. Rather, they reduce the
divergence of folding intermediates into non-productive side reactions.
• Molecular chaperones reversibly bind to and shield unfolded segments of polypeptides that would
otherwise act as loci for aggregation. For in-vitro analysis, it is assumed that protein folding and
unfolding are equivalent.
• Molecular chaperones are often characterized by their ability to prevent aggregation of proteins
unfolded by mild acid or heat treatment. In plant cells, HSP100/Clp is cytoplasmic and organellar
(within plastids and/or mitochondria). HSP100/Clp is expressed in developmental and organ-specific
patterns and is up-regulated by various environmental stress conditions (heat, cold, high salt, and heavy
metals).
• The 90-kD stress proteins, which range in size from 82 to 96 kD, are highly conserved and abundant in
the cytoplasm of both eukaryotic and prokaryotic cells.
• HSP chaperones bind to hydrophobic patches that are exposed to incompletely folded proteins. HSP60
and HSP70 families, help guide the folding of most newly synthesized proteins.
• Hsp60 acts on fully synthesized proteins that have not yet achieved their final folded form. The presence
of incompletely folded proteins can lead to the formation of aggregates that may have dangerous
consequences for the cell.

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Proteasome-mediated protein Degradation

• The ultimate turnover of incompletely folded proteins is mediated by
an abundant ATP-dependent protease, known as the proteasome. The
proteasome is a highly compartmentalized protease with sequestered
active sites.
• The proteasome acts processively to convert the entire protein substrate
into short peptides.
• Proteins destined for degradation are marked by polyubiquitin chains
that are added via a multistep conjugation process.
• The process of degradation can be regulated by altering the activity of
the E3 ubiquitin ligase or the availability of the degradation signal on
the target protein.

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Function of chloroplast with respect to protein

• Chloroplasts are the cellular compartments where the photosynthesis takes place in plant cells. In addition, they are
responsible for producing many of the nutrients that allow the growth of plants and of animals that ingest them.
• A big part of this work is carried out by proteins, some of which are very prone to misfold and aggregate, thus losing their
function. Under normal conditions chloroplasts get rid of these defective proteins by degrading them using the molecular
machinery called protease Clp.
• However, when the accumulation of aggregated proteins exceeds the ability of the Clp protease to remove them, Then
chloroplasts generate a distress signal that travels to the nucleus of the cell to activate the production of repair proteins,
called chaperones.
• The chaperones are transported to the chloroplasts to undo the protein
"lumps" and unfold the disaggregated proteins, favoring that they can be
folded back correctly and recover their function in a few hours.
• The research, conducted with the model plant Arabidopsis thaliana, has
discovered a key gene (HsfA2), which activates the chaperone synthesis and
thus rescues the cell from the toxic effects produced by misfolded protein
accumulations.
HsfA2 gene
• "The signaling pathway from the chloroplasts to the nucleus turns on a
molecular switch called HsfA2. This key gene is also activated when a heat
stroke causes problems of protein folding in other cellular compartments,"

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Incorrect protein folding

• 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.
• 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 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 lead to the formation of amyloid-like
structures which can cause degenerative disorders and cell death.

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Conc…

The problem of misfolding

• When proteins do not fold correctly, their
internal hydrophobic regions become exposed
and interact with other hydrophobic regions on
other molecules, and form aggregates

Outcome of protein misfolding

• Partly folded or misfolded polypeptides or
fragments may sometimes associate with
similar chains to form aggregates.
• Aggregates vary in size from soluble dimers
and trimers up to insoluble fibrillar structures
(amyloid). Improper folding may produce altered protein
(protein misfolding) and disease occurs e.g.
• Both soluble and insoluble aggregates can be Alzheimer and Amyloidosis
toxic to cells

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A Study of Protein
Misfolding in 2013
• The misfolded protein aggregation in plant cells in
response to environmental stresses, including
ultraviolet (UV) radiation, heat stress, and cold
stress.
• Treatment of grape and tobacco cultured cells
with MG-132, a proteasome inhibitor, induced
misfolded protein aggregation.
• Environmental stresses induced the endoplasmic Environmental stresses induce misfolded protein aggregation in plant cells.
reticulum (ER) stress response in the cells. The
cells under ER stress showed aggregation of
misfolded proteins.
• The misfolded protein aggregation was completely
inhibited by treatment of the cells with
trichostatin-A (TA) or colchicine (COL),
suggesting that the misfolded proteins might be
aggregated in plant cells in a microtubule-
dependent manner.
• Detected aggregates were initially observed
immediately after exposure to the environmental
stresses (1 min after UV radiation, 5 min after heat
stress exposure, and 15 min after cold stress
exposure).

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Types of Misfolding
• There are many ways to consider misfolded proteins.
• First, some proteins did not find an alternatively stable conformation and thus misfolded.
• Alternatively, genetic mutations may cause protein misfolding and malfunction; even one erroneous
amino acid can cause an entire protein to fold incorrectly, and The results may include aggregation of
the protein and cellular catastrophe.
• So possible for protein misfolding disorders to arise because of mutations in the cell’s
protein folding machinery. For example, mutations in chaperones would allow proteins
translated to fold into alternative conformations more freely, while mutations in
degradation-related proteins would allow misfolded proteins to escape degradation and
subsequently aggregate and form fibrils.
• Proteins translated from alternatively spliced mRNAs will consequently have differences
in their amino acid sequences and often in their biological functions as well.
• A single nucleotide substitution results in the transformation of a codon for glutamic acid into a codon for valine
and an altered Hemoglobin structure that causes aggregation and formation of fibrils in humans. Some genes can
produce multiple variations of a protein. In these instances, certain exons of a given gene may be included or
excluded from the final processed mRNA produced by that gene. This process is called alternative splicing. An
error that occurs during this splicing process may result in the synthesis of a protein with an incorrectly
incorporated exon, an erroneous amino acid sequence, and ultimately an incorrectly folded protein.

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• In addition to mutation and gene amplification, protein misfolding is also associated with cellular
insults, namely, mitochondrial dysfunction, calcium-induced protein misfolding, and
inflammation.
• Gradual deterioration of mitochondria, which is critical for numerous functions such as oxidative
phosphorylation for ATP production, lipid metabolism, and apoptosis.
• Mitochondrial dysfunction will lead to the overproduction of reactive oxidative species (ROS)
due to irregular oxidative phosphorylation. ROS-mediated insults to proteins may lead to cell
death. Recently, it has been demonstrated that dysfunction of mitochondria-assisted ER
membrane, as well as ER-mitochondria crosstalk, may contribute to a toxic cellular milieu.
• Calcium-induced protein misfolding is initiated by pathological conditions leading to excess
glutamate in the synaptic cleft, resulting in overstimulation of NMDA receptors, which leads to
intracellular calcium influx.
• Excess cytosolic calcium may lead to ROS generation, resulting in nitrosative stress that impairs
quality control mechanisms, thus triggering the accumulation of misfolded proteins.
• Misfolded proteins may establish transmembrane pores that further increase the influx of
calcium, leading to a vicious cycle of cell toxicity.
• Misfolded proteins may also act as irritants, triggering a chronic inflammatory response
involving pro-neurotoxic accumulation of inflammatory proteins in inclusions.

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Conc…
Prion disease
• Striking examples of protein folding-related diseases are prion
diseases, such as Creutzfeldt-Jacob disease (in humans), and mad cow
disease (in cows), and scrapie (in sheep).
• Pathological conditions can result if a brain protein known to as prion
protein (PrPC) is misfolded into an incorrect form called PrPSc due to
mutation in the PRNP gene.
• PrPC has a lot of α-helical conformation, but PrPSc has more β strands
forming aggregates
• The disease is caused by a transmissible agent (Abnormal protein), and
can be acquired by
• Infection PrPC (cellular prion protein) is the normal, non-infectious form
• Inheritance of the prion protein found in the cells of mammals.
• Spontaneously PrPSc (scrapie prion protein) is an abnormal, infectious form of
the prion protein that is associated with prion diseases

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Conc…

Alzheimer’s Disease
• Not transmissible between
individuals
• Extracellular plaques of protein
aggregates of a protein called
tau and another known as
amyloid peptides (Aβ) damage
neurons.

Formation of plaques

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Conc…

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Techniques used to study Protein folding

1. Circular Dichroism:
• Circular Dichroism (CD) is an absorption spectroscopy method based on the differential absorption of left and
right circularly polarized light. Optically active chiral molecules will preferentially absorb one direction of the circularly
polarized light.
• Widely used technique for the study of protein structure.
• This type of spectroscopy measures the absorption of circularly polarized light since the structures of proteins such as the
α-helix and β-sheets are chiral and can absorb this sort of light.
• The peptide backbone forms characteristic secondary structures such as α-helices, β-pleated sheets, turns, and disordered
sections with specific Φ, Ψ dihedral angles and H-bond patterns affecting the CD spectrum.
• Quantitative analysis of CD spectra allows the prediction of the protein secondary structure content. The method is
especially useful when X-ray or NMR techniques fail.
• Absorption of light indicates the degree of protein’s foldedness. Numerous algorithms have been developed for the
estimation of the secondary structure composition from the CD spectra. This technique also measures the equilibrium
unfolding of protein by measuring the change of absorption against denaturant concentration or temperature.
• The denaturant melt measures the free energy of unfolding while the temperature melt measures the melting point of
proteins. This technique is the most general and basic strategy for studying protein folding.

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2. Dual Polarization Interferometry 3. Mass Spectrometry

• Mass spectrometry (MS) is an analytical technique that
separates ionized particles by using differences in the
• A relatively new technique, which explores the molecular
ratios of their charges to their respective masses
layers adsorbed to the surface of a waveguide using an
(mass/charge; m/z).
electromagnetic evanescent wave of a laser beam
• Radar Transmits and receives pulses in both a horizontal
• The advantages of using mass spectrometry to study
and vertical orientation. As a result, the returning
protein folding include the ability to detect molecules
frequencies provide measurements of the horizontal and
with different amounts of deuterium, which allows the
vertical dimensions of targets, supplying forecasters with
heterogeneity of the protein folding reactions to be
better estimates of the size, shape, and variety of targets. studied.
• DPI has been mainly used to study 03 different types of • It can also measure the conformation of folding
interactions: intermediates bound to molecular chaperones without
• Antibody interactions, disrupting the complex.
• Protein−protein interaction, • Mass spectrometry can also directly compare refolding
• Interaction considers the nonprotein ligands. properties, since mixtures of proteins can be studied
without separation if the two proteins have sufficiently
different molecular weights.
• Modern mass spectrometry (MS) technologies have
provided a versatile platform that can be combined with
several techniques to analyze protein structure and
dynamics.

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4. Computational Prediction of Protein Tertiary Structure

• This is a distinct form of protein structure analysis in that it involves protein
folding. These programs can simulate the lengthy folding processes, provide
information on statistical potential, and reproduce folding pathways.
• Both homology modelling (based on sequence comparison) and threading
methods (based on fold-recognition) can be called template-based structure
prediction methods. Unlike homology modelling and threading methods, ab initio
method aims to build structure from the first principles of physics which does not
rely on any previously solved structure. The development of ab initio method is
also the exploration of the second genetic code.

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