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Dr.  Sanjeeva  Srivastava  

IIT  Bombay  

Primary Secondary Tertiary Quaternary

Structure Structure Structure Structure

Polypeptide Assembled
α Helix chain
Amino acid subunits
residues

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• Amino acid sequence determines 3-D structure

• Protein folding
• Thermodynamics of protein folding
• Molecular chaperone for protein folding
• Protein misfolding; diseases

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• Anfinsen’s experiment
• Denaturation
• Refolding

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• Ribonuclease A
• Urea, guanidine HCl
- denaturants
• β-mercaptoethanol
- breaks disulfide bonds

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E R Q H
A K F M D
A A S
1 E T 20 S
K A S T
+H3N N S S S A
30 Y 80
M Q N C
K M I S M T S Y S Q Y
S Q D T
C 70 T C
R R N
E Q C
N
K S S G T G
K
L 90 N A
Y
T 124 V 120 V
P S A D F H V P V Y P N
K N G N
C 110 E
D C K
O O C
R A Q
Y 100 A
C YK T T Q A H I I V S 60
40 K N K C
V
P Q
V A
N V
4 disulfide bonds T F A D
V H E S L
50
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O NH2
+ Cl¯
C C
H 2N NH2 H 2N NH2
Urea Guanidium
Chloride

Effectively disrupts non-covalent

bonds of proteins

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H2
HO C H
C S • Cleavage of disulfide bonds
H2 • Large excess converts disulfides to sulfhydryls
ß-mercaptoethanol
H2
HO C H
C S
H
S H2 S

S S
H
H2 H2
HO C C OH
C S S C
H2 H2

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• 8 M urea and β-mercaptoethanol treatment

- converted native protein to fully reduced,
randomly coiled polypeptide “denatured”
- lacked enzymatic activity

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1 95
H 58
72 S 40
26 S 65
65 H H
84 S
84
110 S
110 8 M Urea & ß- H
95 mercaptoethanol 72 26
12
58 1
40 4

Native Ribonuclease Denatured reduced Ribonuclease

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26 26 72
65
40
58 110 Trace of ß- 84
65 mercaptoethanol
1
110
95 124 95
72
84 58
40
Scrambled Ribonuclease Native Ribonuclease

• Scrambled – wrong pairings, 104

• Trace amount of b-mercaptoethanol catalyzed rearrangement
of disulfide pairing
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• Urea and β-mercaptoethanol removed by dialysis

• Denatured ribonuclease regained activity
• Enzyme refolded into active form
• Sulfhydryl groups became oxidized by air

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Horwich et al. 2002

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• Amino acid sequence determines 3D structure

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Binding site

Protein folding

Unfolded Protein Folded Protein

• Polar amino acid side chains tend to gather on

outside of the protein
• Non-polar amino acid side chains are buried inside

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Hydrophobicity/
Amino Acid Abbreviation Symbol Charge
Aspar&c  acid   Asp   D   Nega&ve  
Glutamic  acid   Glu   E   Nega&ve  
Arginine   Arg   R   Posi&ve  
Lysine   Lys   K   Posi&ve  
His&dine   His   H   Posi&ve  
Asparagine   Asn   N   uncharged  polar  
Glutamine   Gln   Q   uncharged  polar  
Serine   Ser   S   uncharged  polar  
Threonine   Thr   T   uncharged  polar  
Tyrosine   Tyr   Y   uncharged  polar  
Cysteine   Cys   C   non-­‐polar  
Glycine   Gly   G   non-­‐polar  
Isoleucine   Ile   I   non-­‐polar  
Leucine   Leu   L   non-­‐polar  
Methionine   Met   M   non-­‐polar  
Phenylalanine   Phe   F   non-­‐polar  
Proline   Pro   P   non-­‐polar  
Tryptophan   Trp   W   non-­‐polar  
Valine   Val   V   non-­‐polar  
Alanine   Ala   A   non-­‐polar  
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Overall increase in an entropy drives the folding process

Folded conformation in aqueous environment

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• Protein denaturation by chemical and heat

Expose to
high Remove
concentration
Urea
of Urea

Purified Protein Denatured Original

islolated from Protein conformation of
cells protein re-forms

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100
Protein unfolded

0 Denaturant

• A sharp transition from native (folded) to denatured

(unfolded) form
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100
Protein unfolded

50

0 Denaturant

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Many unstable One stable

conformations conformation
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• Folding is a cooperative process

• In general, any protein adopts only one conformation
• Or, few very closely related characteristic functional
conformations “native state”

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• Amino acid sequence dictates protein structure

• knowledge-based and Ab initio “from the
beginning” prediction to predict protein structure

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• Hydrophobic amino acids are driven to associate-

hydrophobic collapse
• Thus, overall increase in an entropy drives the
folding process

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Molten  
globule  
state  

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Molten  
globule  
state  

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• Protein folds into a single, energetically favorable

conformation, specified by its amino acid sequence
• A protein may fold into alternative 3D structure due
to mutations, inappropriate covalent modifications

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Protein
Newly
aggregate
Synthesized
Protein

Correctly Correctly folded Incompletely folded

folded without with help of forms digested by
help molecular proteasome
chaperone

Increasing Time

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• Accumulation of misfolded protein or proteolytic

fragments results into few degenerative diseases
• Characterized by presence of insoluble protein
plaques in organs such as brain and liver

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Fibril axis

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• Anfinsen experiment
• Protein folding
• Thermodynamics of protein folding
• Molecular chaperons
• Protein mis-folding and diseases

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• Horwich A. Protein aggregation in disease: a role for folding

intermediates forming specific multimeric interactions. Volume
110, Issue 9 (November 1, 2002). J Clin Invest. 2002;110(9):
1221–1232. doi:10.1172/JCI16781.
• Harold A. Scheraga. Protein structure and function, from a
colloidal to a molecular view. Carlsberg Research
Communications. January 1984, Volume 49, Issue 1, pp 1-55.
•Berg J., Tmyoczko J. & Stryer L., Biochemitry fifth ed., W. H.
Freeman & company, 2002. ISBN: 0716746840.
•Nelson D. & Cox M.,Lehninger, Principles of Biochemistry fourth
ed., 2004. W. H. Freeman and company. ISBN: 023022699X.
• Creighton T. E., Protein folding first ed., W. H. Freeman and
company, 1992. ISBN: 071677027X.
• Voet D. & Voet J., Biochemistry fourth ed., Wiley, 2000. ISBN:
047158651X.
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