Protein folding and misfolding

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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

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Master Layout (Part 1)
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This animation consists of 5 parts Part 1
Thermodynamics of protein folding Part 2
Anfinsens 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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Entropy
residues in native conformation
Unfolded polypeptide chain
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Free energy
Partially folded polypeptide
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Native state a-helix
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Source Biochemistry by Lehninger, 4th edition
(ebook)
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Definitions of the componentsPart 1
thermodynamics of protein folding
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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
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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
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Unfolded polypeptide chain, high free energy
entropy
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Helix formation commences, free energy entropy
decrease
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Action
Audio Narration
Description of the action
The green chain and blue circles shown must
assume the different arrangements as shown.
(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.
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.
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Source Biochemistry by Lehninger, 4th edition
(ebook)
6
Part 1, Step 2
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Entropy
residues in native conformation
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Intrachain hydrogen bonds
Free energy
Entropy of water molecules increase, and
polypeptide decreases
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Stable native state a-helix
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Action
Audio Narration
Description of the action
The black dotted lines must gradually appear on
the green chain on top. The chain and blue
circles must rearrange themselves.
(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.
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.
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Source Biochemistry by Lehninger, 4th edition
(ebook)
7
Master Layout (Part 2)
1
This animation consists of 5 parts Part 1
Thermodynamics of protein folding Part 2
Anfinsens 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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b-mercaptoethanol
Disulphide bonds
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6M urea
Noncovalent interaction
Remove urea b-mercaptoethanol
Native ribonuclease A
Broken disulphide linkages
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Denatured ribonuclease A
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Definitions of the componentsPart 2
Anfinsens experiment
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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. 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
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b-mercaptoethanol
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6M urea
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Native state ribonuclease A
Broken disulphide linkages
Denatured ribonuclease A
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Action
Audio Narration
Description of the action
The green pie shaped objects must break the black
lines while the blue pie objects must be break
the dotted lines.
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.
Ribonuclease A in its native state has four
disulphide bonds between its cysteine residues.
When treated with b-mercaptoethanol 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
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Denatured ribonuclease A
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Remove urea and b-mercaptoethanol
Remove b-mercaptoethanol only
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Native state ribonuclease A
Inactive ribonuclease A
Action
Audio Narration
Description of the action
The chain must twist itself and reform the
structures shown below. Movement in 3-D must be
shown.
First show the structure on top followed by the
right arrow text. The green pie-shaped 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.
It was observed by Anfinsen that removal of urea
and b-mercaptoethanol 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.
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Master Layout (Part 3)
1
This animation consists of 5 parts Part 1
Thermodynamics of protein folding Part 2
Anfinsens 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
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 componentsPart 3 Amino
acid structure determines 3-D structure
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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. 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
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Protein folding is governed by distribution of
polar non-polar amino acid residues in proteins
Aqueous environment
Polar side chains
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Hydrogen bond interactions
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Non-polar side chains
Folded protein
Hydrophobic residues buried inside
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Action
Audio Narration
Description of the action
Folding of the grey chain.
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.
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
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Protein folding is a cooperative process while
unfolding is a sharp, quick transition
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Unfolded state
Partially folded
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Partially folded conformations
Folded native state
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Action
Audio Narration
Description of the action
Show the figures above appearing one at a time
followed by the graph.
(Please redraw all figures.)First show each of
the figures appearing one after another. In each,
the chain must become more compact as it
progresses. Once the last figure has been shown,
the graph must appear with the blue figure being
unfolded to give the red chain. As soon as the
structure starts getting modified, there must be
a rapid increase in the graph curve.
Proteins typically adopt only one characteristic
functional native state conformation which has
lowest free energy and is most stable. Folding is
limited to one conformation due to properties of
the amino acid side chains such as
hydrophobicity, size, shape etc. Folding is a
highly cooperative process wherein there is
progressive stabilization of the intermediates.
Although it is theoretically possible to predict
protein structure from the amino acid sequence,
several long-range interactions often limit these
predictions.
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Master Layout (Part 4)
1
This animation consists of 5 parts Part 1
Thermodynamics of protein folding Part 2
Anfinsens 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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Source Biochemistry by Lehninger, 4th edition
(ebook)
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Definitions of the componentsPart 4 Molecular
chaperones for protein folding
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1. Unfolded protein This refers to the protein
or polypeptide chain that has not been folded or
is in a partially folded state. 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). 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
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DnaJ
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Unfolded protein
DnaK
Pi
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Action
Audio Narration
Description of the action
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.
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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Source Biochemistry by Lehninger, 4th edition
(ebook)
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Part 4, Step 2
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GrpE
2
3
DnaJ
DnaK
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Action
Audio Narration
Description of the action
(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.
The green pie objects must remove the grey
circles from the attached rectangle.
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.
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Source Biochemistry by Lehninger, 4th edition
(ebook)
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Part 4, Step 3
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2
DnaK regenerated for next round of protein folding
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Partially folded protein
DnaK
Folded native protein
To GroEL system
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Action
Audio Narration
Description of the action
(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.
The red ribbon must detach from purple square
which must again bind the yellow circle.
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.
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Source Biochemistry by Lehninger, 4th edition
(ebook)
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Master Layout (Part 5)
1
This animation consists of 5 parts Part 1
Thermodynamics of protein folding Part 2
Anfinsens experiment Part 3 Amino acid
structure determines 3-D folding Part 4
Molecular chaperones for protein folding Part 5
Protein misfolding diseases
Alzheimers disease
CreutzfeldtJakob disease
Huntingtons disease
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Cystic fibrosis
Pulmonary emphysema
Lathyrism
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Definitions of the componentsPart 5 Protein
misfolding diseases
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• Alzheimers Disease
• 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
  Alzheimers 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. Huntingtons disease
• 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 componentsPart 5 Protein
misfolding diseases
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• 3. CreutzfeldtJakob 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 componentsPart 5 Protein
misfolding diseases
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• 5. Pulmonary emphysema
• 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
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Action
Audio Narration
Description of the action
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.
ltAs given in the definitions slides.gt
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Interactivity option 1Step No1
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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?
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a) UV spectroscopy
b) NMR
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c) ESR
d) Mass spectroscopy
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Results
Boundary/limits
Interacativity Type 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.
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.
Choose the correct option.
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Interactivity option 1Step No2
Deuterium labelled peptide N atoms
Urea
D2O
Guanidinium chloride
Denatured protein with deuterated peptide
nitrogen atoms.
Native protein with regular hydrogen atoms
Stopped -flow device
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Interactivity option 1Step No3
Folding initiated in denatured protein by mixing
with denaturant diluted H2O and decreasing pH to
arrest exchange reaction.
Folding takes place for preset time t, after
which pH is rapidly increased again for exchange
reaction to occur.
Deuterium label
Folding for time t
Not involved in H-bonding
Low pH no exchange reaction
Only those D atoms that have not been involved in
hydrogen bonding by time t will get exchanged now.
Denatured protein
pH increased
Regular hydrogen from H2O
H-D exchange reaction
pH lowered finally to terminate labelling pulse
and H/D ratio at each exchangeable site
determined by 2-D NMR.
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Questionnaire

• 1. If the free energy of a reaction is negative,
  the reaction will be
• 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
• 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. Which of the following is a trinucleotide
  repeat disorder?
• Answers a) Cystic fibrosis b) Alzheimers
  c) Huntingtons d)? Lathyrism
• 5. Which enzyme is inactivated in the disease
  lathyrism?
• Answers a) Hexokinase b) Lysyl oxidase c)
  Collagenase d)? Prolyl hydroxylase

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Links for further reading
Books Biochemistry by Stryer et al., 56th
edition Biochemistry by A.L.Lehninger et al., 4th
edition Biochemistry by Voet Voet, 3rd edition