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Protein folding and misfolding
Folding of proteins into their native conformations
occurs spontaneously under physiological conditions
and is dictated by the primary structure of the protein.
Harini Chandra
Affiliations
Master Layout (Part 1)
1
This animation consists of 5 parts:
Part 1 – Thermodynamics of protein folding
Part 2 – Anfinsen’s experiment
Part 3 – Amino acid structure determines 3-D folding
Part 4 – Molecular chaperones for protein folding
Part 5 – Protein misfolding diseases
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3
Partially folded
polypeptide
4
Native state
a-helix
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Source: Biochemistry by Lehninger, 4th edition (ebook)
Free energy
Unfolded polypeptide chain
% residues in native conformation
Entropy
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Definitions of the components:
Part 1 – thermodynamics of protein folding
1. Entropy: Entropy is a measure of randomness or how disorganized a
system is and forms the basis of the second law of thermodynamics, which
states that the total entropy of a system cannot decrease without
correspondingly increasing the entropy of another system. In other words,
the entropy of the universe (system + surrounding) is constantly increasing.
Entropy helps in predicting the spontaneity of any process. An unfolded
polypeptide chain has high entropy which goes on decreasing as the protein
folds into its native state.
2. Free energy: The free energy, also known as Gibbs free energy, is the
maximum amount of mechanical work that can be done by a system at
constant temperature and pressure. In general, all systems try to attain
minimum free energy and a reaction takes place spontaneously only when
the associated free energy change is negative.
3. Unfolded polypeptide chain: The amino acids that have been joined
together by peptide bonds but have not yet formed their secondary or
tertiary structures. This conformation has the highest free energy and
entropy.
4. Partially folded polypeptide: The amino acids in the polypeptide chain
start interacting by means of hydrogen bonds across the polypeptide
backbone in order to initiate the folding process. The free energy and
entropy of the system gradually decrease as the folding takes place.
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Definitions of the components:
Part 1 – thermodynamics of protein folding
5. Native state a-helix: The polypeptide chain assumes its most stable,
native conformation in the form of an a-helix with the folding being directed
largely by its amino acid residues. This conformation corresponds to
minimum free energy and entropy, thereby conferring very high stability. The
lowering of entropy is favoured by a corresponding increase in entropy in the
surroundings composed of water molecules.
6. Molten globule: Initial collapsed state of a protein with very little
thermodynamic stability is known as the molten globule. The amino acid side
chains are extremely disordered in this state with several fluctuations being
observed.
7. Percentage residues in native conformation: This refers to the number
of residues that have assumed their favourable, lowest energy states. The
percentage increases gradually as the folding process takes place.
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Part 1, Step 1:
H2O
H2O
H2O
H2O
Beginning of helix formation
2
H2O
H2O
Unfolded polypeptide chain, high
free energy & entropy
H2O
H2O
H2O
H2O
4
H2O
Helix formation commences, free
energy & entropy decrease
Action
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Free energy
3
H2O
Entropy
The green
chain and blue
circles shown
must assume
the different
arrangements
as shown.
Description of the action
(Please redraw all figures.)
First show the green chain on the top. Then show
the blue circles appearing around it .
The green chain must then be shown to bend in
various ways until it assumes the shape below. And
the blue circles must also rearrange themselves as
shown. The graph on the right must gradually take
shape in the direction indicated.
Source: Biochemistry by Lehninger, 4th edition (ebook)
Audio Narration
An unfolded polypeptide chain has very
high free energy and entropy. Protein
folding acts to decrease the free energy
of the system by forming favorable
interactions and assuming a more stable
state. The entropy of the polypeptide
chain decreases during this process.
Part 1, Step 2:
H2O H2O
H2O H2O
H2O
Entropy
Intrachain hydrogen
bonds
H2O
H2O H O
2
Entropy of water molecules increase,
and polypeptide decreases
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H2O
H2O
H2O H O
2
H2O H O
2
H2O
H2O H O
2
H2O H O
2
Stable native
state a-helix
4
Action
5
Free energy
2
% residues in native conformation
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The black dotted
lines must
gradually appear
on the green
chain on top. The
chain and blue
circles must
rearrange
themselves.
Description of the action
(Please redraw all figures.)
The blue circles must move around away from
the green chain on top and form small
clusters.
The black dotted lines must appear on as
shown on the green chain on top.
The figure below must appear as shown and
the graph on the right must be gradually
completed.
Source: Biochemistry by Lehninger, 4th edition (ebook)
Audio Narration
As the protein continues to fold in order to assume its
stable, low energy native state conformation, the entropy
also decreases. While this would seem unfavorable for the
system, it must be recalled that the entropy of the
surrounding water molecules increases during the process,
thereby increasing the overall entropy and making it
favorable and spontaneous.
Master Layout (Part 2)
1
2
This animation consists of 5 parts:
Part 1 – Thermodynamics of protein folding
Part 2 – Anfinsen’s experiment
Part 3 – Amino acid structure determines 3-D folding
Part 4 – Molecular chaperones for protein folding
Part 5 – Protein misfolding diseases
b-mercaptoethanol
Disulphide
bonds
3
6M urea
Noncovalent
interaction
Native ribonuclease A
Remove urea &
b-mercaptoethanol
Broken
disulphide
linkages
4
Denatured ribonuclease A
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2
3
Definitions of the components:
Part 2 – Anfinsen’s experiment
1. Native ribonuclease A: This is an endonuclease enzyme composed of
124 amino acids that cleaves single-stranded RNA molecules. It has four
disulphide bonds in its native state that are essential for conformational
folding and enzymatic activity. This was used by Christian Anfinsen to
postulate the thermodynamic hypothesis of protein folding, according to
which the folded form of a protein represents its free energy minimum.
2. b-mercaptoethanol: b or 2-mercaptoethanol with the formula
OHCH2CH2SH is a chemical compound that is used commonly to reduce
disulphide linkages in proteins, thereby disrupting the tertiary and quaternary
structures.
3. 6M urea: It is an organic compound having two amine groups joined by a
carbonyl group and used at concentrations up to 10 M for denaturing proteins
by breaking the noncovalent interactions.
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4. Denatured ribonuclease A: On treatment with b-mercaptoethanol and
urea, the ribonuclease A loses its native conformation due to breaking of the
disulphide and noncovalent linkages. Activity of the enzyme is also lost during
this process. However, it was observed by Anfinsen that removal of both urea
and b-mercaptoethanol allows the enzyme to fold into its native conformation
again with more than 90% enzymatic activity.
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Part 2, Step 1:
b-mercaptoethanol
2
6M urea
3
Native state ribonuclease A
Broken disulphide
linkages
Denatured ribonuclease A
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Action
5
The green pie
shaped objects
must break the
black lines while
the blue pie
objects must be
break the dotted
lines.
Description of the action
First show the structure on the left. Then
show the green pie shaped objects moving
towards the black lines and breaking them
and simultaneously the blue pie shaped
objects breaking the dotted lines.
The structure must then unfold and give rise
to the open chain displayed below with the
green & blue objects remaining bound to it.
Audio Narration
Ribonuclease A in its native state has four
disulphide bonds between its cysteine
residues. When treated with bmercaptoethanol and 6M urea, the protein
undergoes denaturation and the disulphide
linkages are broken. Enzyme activity is lost
in the denatured state.
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Part 2, Step 2:
Denatured ribonuclease A
2
Remove urea and
b-mercaptoethanol
Remove bmercaptoethanol only
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4
Native state ribonuclease A
Action Description of the action
5
The chain must
twist itself and
reform the
structures
shown below.
Movement in 3D must be
shown.
First show the structure on top followed
by the right arrow & text. The green pieshaped objects must be removed & the
structure on the right must appear. Next
the left arrow & text must be shown with
disappearance of the blue pie-shaped
objects & appearance of the figure on
the left.
Inactive ribonuclease A
Audio Narration
It was observed by Anfinsen that removal of urea and bmercaptoethanol led to the refolding of the enzyme to
assume its native state with more than 90% enzyme
activity being intact. However, if only b-mercaptoethanol
was removed in presence of urea, the formation of
disulphide bonds was random, leading to enzyme with
only around 1% activity.
Master Layout (Part 3)
1
This animation consists of 5 parts:
Part 1 – Thermodynamics of protein folding
Part 2 – Anfinsen’s experiment
Part 3 – Amino acid structure determines 3-D folding
Part 4 – Molecular chaperones for protein folding
Part 5 – Protein misfolding diseases
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Protein folding
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Amino acid sequence 1
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2
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Protein 1
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Amino acid sequence 2
Protein 2
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Source: Biochemistry by Stryer, 5th edition (ebook)
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Definitions of the components:
Part 3 – Amino acid structure determines 3-D
structure
1. Amino acid sequence 1, 2: These are two completely different amino
acid sequences that will give rise to different protein structures.
2. Protein 1, 2: The protein structure corresponding to amino acid sequence
1 and 2 respectively. The first amino acid sequence cannot give rise to the
second protein structure & vice versa.
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3. Protein folding: The process by which the amino acid side chains in the
proteins interact with one another to form energetically favourable bonds
with each other thereby allowing regions that are far away from one another
to move closer. This process is determined by the amino acid sequence of
the proteins and needs to be energetically feasible in order to take place.
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Part 3, Step 1:
Protein folding is governed by distribution of polar &
non-polar amino acid residues in proteins
Aqueous environment
Polar side chains
H2O
H2O
2
H2O
H2O
Hydrogen bond
interactions
Non-polar side
chains
3
Folded protein
Hydrophobic
residues buried
inside
H2O
H2O
H2O
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Action Description of the action
Folding of
the grey
chain.
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First show the structure on the left with the
blue & green projections. Next show this
chain folding such that all the green parts
come on the inside & blue parts are on the
outside as shown in figure on the right. This
must be surrounded by water molecules
which must move towards the blue regions as
depicted in the animations.
Audio Narration
The process of protein folding is governed by the distribution of
polar and non-polar amino acid residues in the protein.
Hydrophobic amino acids are driven to interact with one
another, a process termed as hydrophobic collapse. They come
together andin the process, eliminate water molecules around
them. The polar residues remain on the surface and form
hydrogen bonds with water molecules while the hydrophobic
residues get buried within the core of the protein.
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Part 3, Step 2:
Protein folding is a cooperative process while unfolding is a sharp, quick transition
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Unfolded state
3
Partially folded
Partially folded
conformations
Folded native state
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Action Description of the action
5
Show the
figures
above
appearing
one at a
time
followed by
the graph.
Audio Narration
Proteins typically adopt only one characteristic functional native state
(Please redraw all figures.)First show
conformation which has lowest free energy and is most stable.
each of the figures appearing one after
another. In each, the chain must become Folding is limited to one conformation due to properties of the amino
more compact as it progresses. Once the acid side chains such as hydrophobicity, size, shape etc. Folding is a
highly cooperative process wherein there is progressive stabilization
last figure has been shown, the graph
of the intermediates. Although it is theoretically possible to predict
must appear with the blue figure being
unfolded to give the red chain. As soon as protein structure from the amino acid sequence, several long-range
the structure starts getting modified, there interactions often limit these predictions.
must be a rapid increase in the graph
curve.
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Master Layout (Part 4)
This animation consists of 5 parts:
Part 1 – Thermodynamics of protein folding
Part 2 – Anfinsen’s experiment
Part 3 – Amino acid structure determines 3-D folding
Part 4 – Molecular chaperones for protein folding
Part 5 – Protein misfolding diseases
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2Pi
DnaJ
Unfolded protein
3
DnaK
To GroEL
system
4
Folded native
conformation
protein
Partially folded
protein
GrpE
ADP + GrpE + DnaJ
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Source: Biochemistry by Lehninger, 4th edition (ebook)
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Definitions of the components:
Part 4 – Molecular chaperones for protein folding
1. Unfolded protein: This refers to the protein or polypeptide chain that has
not been folded or is in a partially folded state.
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2. DnaJ and Dna K: These are molecular chaperones found in E.coli that are
analogous to the eukaryotic heat shock protein (Hsp) chaperone system.
These chaperones are proteins that interact with unfolded or partially folded
proteins and provide them with suitable microenvironments in which folding
can occur. In addition to this chaperone system, the Hsp proteins have also
been studied and have been found in abundance in cells that have been
stressed by elevated temperatures.
3. ATP: Adenosine triphosphate (ATP) is the energy currency of the cell due
to its high energy phosphate bonds. It gets hydrolyzed to liberate adenosine
diphosphate (ADP) and a phosphate group (Pi).
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4. GrpE: This is a nucleotide exchange factor present in bacterial systems
that facilitates the release of bound ADP.
5. GroEL system: The GroEL system refers to another group of elaborate
protein complexes known as chaperonins that assist the folding of several
cellular proteins.
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Part 4, Step 1:
ATP
DnaJ
2
ATP
Unfolded protein
ATP
DnaK
ATP
3
ADP
ADP
4
Action Description of the action
5
Pi
Blue squares
and purple
figures must
bind to the red
ribbon. After
this, colour of
circle must
change as
shown below.
(Please redraw all figures.)
First show the red ribbon binding to the
blue squares followed by purple figure
(DnaK). The blue squares must interact
with the circles which must then
change colour as shown in the bottom
figure and the arrow stemming out of
the downward arrow must appear.
th
Audio Narration
The unfolded protein is bound by DnaJ and then by
DnaK which is an ATP bound protein. The hydrolysis of
ATP into ADP and Pi by DnaK is stimulated by DnaJ.
The resulting DnaK-ADP remains tightly bound to the
unfolded protein.
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Part 4, Step 2:
ADP
ADP
GrpE
2
ADP
ADP
ADP
3
ADP
DnaJ
DnaK
4
Action Description of the action
5
The green pie
objects must
remove the
grey circles
from the
attached
rectangle.
(Please redraw all figures.)
First show the figure on top left. Then show the
green pie shapes moving to the grey circles such
that they must remove them from the attached
rectangle.
The blue squares must also get detached
resulting in the figure at the bottom.
Source: Biochemistry by Lehninger, 4th edition (ebook)
Audio Narration
The nucleotide exchange factor GrpE present
in bacteria facilitates release of ADP along with
DnaJ. This leaves the DnaK bound to the
partially folded protein which continues to
undergo folding to a more favorable low energy
conformation.
1
Part 4, Step 3:
2
ATP
ATP
ATP
Partially folded
protein
3
DnaK
Folded native
protein
To GroEL
system
4
Action Description of the action
5
DnaK
regenerated for
next round of
protein folding
The red ribbon
must detach
from purple
square which
must again
bind the yellow
circle.
(Please redraw all figures.)
First show the red ribbon being
detached from the purple squares
followed by binding of yellow circles to
the purple squares.
Audio Narration
Once the protein gets completely folded, it gets
detached from DnaK which then binds ATP again,
thereby completing the cycle and preparing it for the
next round of protein folding. Any protein which may
not have been folded completely is then taken over by
the GroEL chaperonin system which completes the
folding.
Source: Biochemistry by Lehninger, 4th edition (ebook)
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Master Layout (Part 5)
This animation consists of 5
parts:
Part 1 – Thermodynamics of
protein folding
Part 2 – Anfinsen’s experiment
Part 3 – Amino acid structure
determines 3-D folding
Part 4 – Molecular chaperones
for protein folding
Part 5 – Protein misfolding
diseases
Alzheimer’s
disease
Huntington’s
disease
Creutzfeldt–
Jakob disease
Cystic fibrosis
Pulmonary
emphysema
Lathyrism
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Definitions of the components:
Part 5 – Protein misfolding diseases
1. Alzheimer’s Disease:

2




3
Structure of certain normal soluble cellular proteins normally rich in alpha helical
regions converted into beta strand conformations which further link with each other
to form beta sheet aggregates known as amyloids.
Insoluble amyloid plaques are essentially made up of a single polypeptide chain or
fibrils known as amyloid-b-protein (Ab).
Observed in the brain of patients with Alzheimer’s where dead or dying neurons
surround plaques.
Neurotoxicity believed to be caused by the Ab fibrils before they get deposited as
amyloid plaques.
The disease presents various symptoms such as memory loss, decreased
neuromuscular coordination, confusion and dementia.
2. Huntington’s disease:


4




5
Neurodegenerative disorder of genetic origin affecting muscular coordination.
Caused by increased number of trinucleotide repeats, CAG, in Huntingtin gene
leading to increased number of glutamine residues incorporated in corresponding
protein.
This alters the folding of the Huntington protein which has highest concentration in
brain and testes.
Exact function of the protein is unclear but is known to interact with several other
proteins.
Mutated protein has also been found to have effects on chaperone proteins which in
turn help in folding several other proteins.
Prominently affects basal ganglia which plays a key role in movement and
behavioural control.
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Definitions of the components:
Part 5 – Protein misfolding diseases
3. Creutzfeldt–Jakob disease:
Initially believed to be caused by viruses or bacteria.
Later discovered to be transmitted by small proteins known as prions.
Prion proteins composed of beta sheet structures that have been modified from
previously existing alpha helices.
Protein aggregates of one abnormal protein sufficient to function as a nuclei for other
normal proteins to attach themselves to.
Characterized by muscular spasms, loss of muscle control and memory loss.
4. Cystic fibrosis:
Autosomal recessive disorder caused by a mutation in gene for the protein cystic
fibrosis transmembrane conductance regulator (CFTR) .
CFTR regulates components of sweat, digestive juices and mucus.
Caused by a deletion of three nucleotides leading to the elimination of a phenylalanine
residue from the protein and therefore abnormal folding.
Dysfunctional protein gets degraded by the cell.
Disorder can affect several body parts such as the lungs, GI tract and reproductive
organs.
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Definitions of the components:
Part 5 – Protein misfolding diseases
5. Pulmonary emphysema:
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3
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Progressive disease of the lung causing shortness of breath.
Can be caused by deficiency of the protein alpha-1-antitrypsin (A1AT).
A1AT is responsible for protecting the lung tissues from damage by enzyme
neutrophil elastase.
Abnormally secreted A1AT gets accumulated in the liver thereby allowing lung tissue
damage.
Causes wheezing, shortness of breath, asthma-like symptoms and also liver cirrhosis.
6. Lathyrism:
Regular ingestion of seeds from sweet pea (Lathyrus odoratus) causes disruption of
cross-linking in the muscle protein, collagen.
Collagen is an important structural protein having a triple helical structure.
Cross-links formed are due to the oxidation of lysine residues by the enzyme lysyl
oxidase to form allysine.
These are essential for proper folding of collagen, giving it the required strength.
b-aminopropionitrile, present in abundance in sweet pea, deactivates this enzyme
by binding to its active site
This prevents cross-linking and proper folding of the protein.
Causes muscle fragility and weakness.
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Part 5, Step 1:
Alzheimer’s
disease
Huntington’s
disease
Creutzfeldt–
Jakob
disease
Cystic
fibrosis
2
Pulmonary
emphysema
Lathyrism
3
4
Action Description of the action
5
User should be
allowed to click
on any of the
given labels to
understand
more about it.
User should be allowed to click
on any of the given labels to
understand more about it as
given by the definitions in the
previous three slides.
Audio Narration
<As given in the definitions slides.>
1 Interactivity option 1:Step No:1
2
3
Protein folding is an extremely quick process with a time scale of few milliseconds to seconds. Monitoring
the process is therefore a formidable task requiring very rapid measurements to be made. One of the
suitable techniques for this measurement is the pulsed hydrogen-deuterium (H/D) exchange reaction.
This relies on the differential measurement of hydrogen and deuterium which give different signals by a
particular spectroscopic technique. Which technique is this?
a) UV spectroscopy
b) NMR
c) ESR
d) Mass spectroscopy
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Interacativity Type
5
Choose the correct
option.
Options
User has to choose one of
the four options. If a, c or d
are chosen, they must turn
red. User can however
continue till he gets the
right answer (b) which must
turn green. User is then
directed to step 2.
Boundary/limits
Results
User has to choose one of the
four options. If a, c or d are
chosen, they must turn red.
User can however continue till
he gets the right answer (b)
which must turn green. User is
then directed to step 2.
Interactivity option 1:Step No:2
Deuterium labelled
peptide N atoms
Urea
D2O
Guanidinium
chloride
Denatured protein
Native protein with
with deuterated
regular hydrogen
peptide nitrogen
atoms
atoms.
Light source
Stopping
syringe
Denatured
protein solution
Mixer
Denaturant
solution with H2O
Detector
Stopped -flow device
Computer
Stop
switch
Interactivity option 1:Step No:3
Light source
Stopping
syringe
Denatured
protein solution
Stop
switch
Folding initiated in denatured
protein by mixing with denaturant
diluted H2O and decreasing pH to
arrest exchange reaction.
Mixer
Denaturant
solution with H2O
Detector
Deuterium label
Computer
Folding for
time ‘t’
Not involved in
H-bonding
Low pH – no
exchange
reaction
Denatured protein
pH increased
Regular hydrogen
from H2O
Folding takes place for preset time
t, after which pH is rapidly
increased again for exchange
reaction to occur.
Only those D atoms that have not
been involved in hydrogen
bonding by time t will get
exchanged now.
H-D exchange reaction
pH lowered finally to terminate
labelling pulse and H/D ratio at
each exchangeable site
determined by 2-D NMR.
1
Questionnaire
1. If the free energy of a reaction is negative, the reaction will be
2
Answers: a) Endothermic b) Exothermic c) Non-spontaneous d) Spontaneous
2. b-mercaoptoethanol is responsible for breaking which type of interactions in proteins?
Answers: a) Disulphide b) Hydrophobic c) Hydrogen bonds d) Peptide bond
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3. In the absence of b-mercaoptoethanol but presence of urea what happens to a denatured
protein?
Answers: a) It gets further denatured b) Random disulphide links are formed c) The native
state is restored d) Only non-covalent interactions are restored
4
4. Which of the following is a trinucleotide repeat disorder?
Answers: a) Cystic fibrosis b) Alzheimer’s c) Huntington’s d) Lathyrism
5. Which enzyme is inactivated in the disease lathyrism?
5
Answers: a) Hexokinase b) Lysyl oxidase
c) Collagenase d) Prolyl hydroxylase
Links for further reading
Books:
Biochemistry by Stryer et al., 5&6th edition
Biochemistry by A.L.Lehninger et al., 4th edition
Biochemistry by Voet & Voet, 3rd edition