Protein structure- Four levels of the organization of protein structure; Primary, secondary, tertiary and quaternary protein structure.

1. Protein structure
and
Protein unfolding & misfolding
Namrata Chhabra
MHPE, FAIMER FELLOW, MD, MBBS

2. Introduction
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4. Levels of organization of Protein structure
• Primary
• Secondary
• Tertiary, and
• Quaternary
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5. Primary structure
Primary structure simply denotes the number and specific
sequence of amino acids in the protein.
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6. Primary structure
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7. Peptide bond
The amino acids in a peptide
chain are linked together by
a peptide bond
A peptide bond is a special type of amide bond formed between two
molecules where an α-carboxyl group of one molecule reacts with the
α-amino group of another molecule releasing a water molecule.
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8. Characteristics of a peptide bond
• Covalent bond
• Stable
• Rigid
• Partial double bond
• Anhydrous
• Uncharged
• Planar
• Trans
• Degradation takes place through
hydrolysis
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9. Peptide bond
The peptide bond is also referred to as the isopeptide bond where the amide bond
forms between the carboxyl group of one amino acid and the amino group of
another amino acid at other positions than the alpha.
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10. Peptide structure
Backbone
Positive
terminal
Negative
terminal
Side chains-Variable groups
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11. The primary structure determines the higher levels
of protein organization
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12. Primary structure
The sequence of amino acids is guided by the genetic information present on
the DNA.
A single nucleotide change in the DNA can bring about alteration in the amino
acid sequence with the resultant loss of partial or complete loss of functional
capacity of the protein.
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13. Primary structure
A single nucleotide change in the genetic information for the synthesis of Beta globin
chain of Hemoglobin results in the misincorporation of valine instead of glutamic acid
in sickle cell anemia causing gross alterations in the oxygen carrying capacity of
hemoglobin.
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14. Protein structure
A hemoglobin molecule is made up of
two α chains and two β chains, each
consisting of about 150 amino acids, for
a total of about 600 amino acids in the
whole protein.
The difference between a normal
hemoglobin molecule and a sickle cell
molecule is just 2 amino acids out of the
approximately 600.
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15. Primary structure
• The glutamic acid-to-valine
amino acid change makes the
hemoglobin molecules
assemble into long fibers.
• The fibers distort disc-shaped
red blood cells into crescent
shapes.
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17. Secondary
structure
Secondary structure is formed by the folding of short
contiguous segments of polypeptide into geometrically
ordered units.
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18. Alpha helix
• The polypeptide backbone of an α helix is
twisted by an equal amount about each α -
carbon.
• A complete turn of the helix contains an
average of 3.6 aminoacyl residues, and the
distance it rises per turn (its pitch) is 0.54 nm.
• The R groups of each aminoacyl residue in an
α helix face outward
• Proteins contain only L-amino acids, for which
a right-handed helix is by far the more stable,
and
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19. Alpha helix
Alpha helix
• The stability of a helix arises primarily
from hydrogen bonds formed between
the oxygen of the peptide bond
carbonyl and the hydrogen atom of the
peptide bond nitrogen of the fourth
residue down the polypeptide chain.
• This pattern of bonding pulls the
polypeptide chain into a helical
structure that resembles a curled
ribbon.
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20. Amino acids that disrupt the alpha helical
structure
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21. Proteins containing alpha helical structure
Hemoglobin, myoglobin and alpha keratin proteins have most of their
structure organized in the form of alpha helix.
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22. Secondary structure- Beta pleated sheets
The second (hence "beta") recognizable regular
secondary structure in proteins is the β sheet.
The amino acid residues of a β sheet, when
viewed edge-on, form a zigzag or pleated
pattern in which the R groups of adjacent
residues point in opposite directions.
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23. Secondary structure- Beta pleated sheets
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24. Secondary structure- Beta pleated sheets
Interacting sheets can be arranged
either to form a parallel β sheet, in
which the adjacent segments of the
polypeptide chain proceed in the
same direction amino to carboxyl,
or an antiparallel sheet, in which
they proceed in opposite directions.
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25. Alpha helix versus Beta pleated sheets
• Unlike the compact backbone of the
helix, the peptide backbone of the
sheet is highly extended.
• But like the helix, sheets derive
much of their stability from hydrogen
bonds between the carbonyl oxygens
and amide hydrogens of peptide
bonds.
• However, in contrast to the helix,
these bonds are formed with
adjacent segments of sheet.
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26. Proteins with Beta pleated sheets
Beta keratin Silk Fibroin
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27. Beta bends, loops and coils
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28. Beta turns and bends
• Short segments of amino acids that join two units
of secondary structure, such as two adjacent
strands of an antiparallel sheet.
• A turn involves four aminoacyl residues, in which
the first residue is hydrogen-bonded to the fourth,
resulting in a tight 180-degree turn.
• Proline and glycine often are present in turns.
• The other amino acids are charged amino acids.
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29. Loops and coils
• Loops are regions that contain residues beyond the minimum number
necessary to connect adjacent regions of secondary structure.
• For many enzymes, the loops bridge substrate binding and catalytic
domains
• Loops are stabilized through hydrogen bonding, salt bridges, and
hydrophobic interactions with other portions of the protein.
• Coils are longer than the loops and connect the adjacent secondary
structures.
• Proteins may contain certain "disordered" regions, often at the extreme
amino or carboxyl terminal ends characterized by high conformational
flexibility.
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30. Super secondary structures
• Globular proteins are constructed by combining secondary structural
elements.
• These form primarily the core region and are connected by loop
regions at the surface of the protein.
• Super secondary structures are usually formed by packing side chains
from adjacent structural elements close to each other.
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31. Super-secondary structures
Beta – alpha –beta, Beta meanders and Greek key are the
super secondary structures
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32. Tertiary structure
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33. Tertiary structure
“Tertiary structure" refers to the entire three-dimensional
conformation of a polypeptide.
It indicates, in three-dimensional space, how secondary structural
features—helices, sheets, bends, turns, and loops—assemble to form
domains and how these domains relate spatially to one another.
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34. Domain
A domain is a section of protein structure sufficient to perform a
particular chemical or physical task such as binding of a substrate or
other ligand.
Other domains may anchor a protein to a membrane or interact with a
regulatory molecule that modulates its function.
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35. Forces stabilizing tertiary structure
• Higher orders of protein structure are stabilized primarily—and often
exclusively—by noncovalent interactions.
• Principal among these are hydrophobic interactions that drive most
hydrophobic amino acid side chains into the interior of the protein,
shielding them from water.
• Other significant contributors include hydrogen bonds and salt
bridges between the carboxylates of aspartic and glutamic acid and
the oppositely charged side chains of protonated lysyl, argininyl, and
histidyl residues.
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36. Forces stabilizing tertiary structure
• While individually weak, collectively these
numerous interactions confer a high degree of
stability to the biologically functional
conformation of a protein.
• Some proteins contain covalent disulfide (S—S)
bonds that link the sulfhydryl groups of
cysteinyl residues. Formation of disulfide bonds
involves oxidation of the cysteinyl sulfhydryl
groups and requires oxygen.
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37. Case study
A 16-year-old girl reported to the dermatologist with a minor scalp
injury. History revealed that all this occurred
after a visit to her hair stylist. Her hair dresser used rollers and applied
some lotion to create permanent waves
for her hair. Immediately after that she started having burning
sensation and itching in the scalp.
On local examination, the scalp was found to be red, tender and a little
ulcerated.
It was a minor allergic reaction to the styling lotion.
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38. Case study
A local ointment and antibiotic supplementation was given and the girl
was sent back home.
What is the basis for permanent hair waving?
Is the permanent wave truly permanent?
What is the nature of the solution used for permanent hair waving?
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39. Hair waving
• A hair is an array of many α-
keratin filaments.
• When hair is exposed to moist
heat, it can be stretched.
• At molecular level, the α-keratin
of hair are stretched out until they
arrive at the fully extended β
conformation.
• On cooling they spontaneously
revert to the α-helical
conformation.
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40. Permanent hair waving
• The characteristic “stretchability” of α-keratins, as well as numerous
disulfide cross linkages, are the basis of permanent waving.
• The hair to be waved or curled is first bent around a form of
appropriate shape.
• A solution of a reducing agent, thiol or sulfhydryl group (-SH), is then
applied with heat.
• The reducing agent then cleaves the cross-linkages by reducing each
disulfide bond to form two cys residues.
• The moist heat breaks hydrogen bonds and causes the α-helical
structure of the polypeptide chains to uncoil.
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41. Permanent hair waving
• After a time, the reducing solution is removed, and an oxidizing agent
is added to establish new bond between pairs of cys residues of
adjacent polypeptide chains, but not the same pairs as before the
treatment.
• After the hair is washed and cooled, the polypeptide chains revert to
there α-helical conformation.
• The hair fibers now curl in the desired fashion because the new
disulfide cress-linkage exert same torsion or twist on the bundle of α-
helical coils in the hair fibers.
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42. Quaternary
structure
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43. Quaternary structure
• Quaternary structure defines the polypeptide composition of a
protein and, for an oligomeric protein, the spatial relationships
between its subunits or protomers.
• Monomeric proteins consist of a single polypeptide chain.
• Dimeric proteins contain two polypeptide chains.
• Homodimers contain two copies of the same polypeptide chain, while
in a heterodimer the polypeptides differ.
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44. Quaternary structure
Greek letters ( α,β,ϒ,δ etc) are used to distinguish different subunits of
a hetero oligomeric protein, and subscripts indicate the number of
each subunit type.
Examples- Immuno globulins are composed of 4 polypeptide chains,
two light chain and two heavy chains , the enzyme Lactate
dehydrogenase(LDH) has two types of polypeptide chains arranged in
the form of a tetramer, CPK(Creatine phosphokinase) enzyme has two
polypeptide chains in its structure.
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45. Lactate dehydrogenase
Structure of
Immunoglobulin
Creatine kinase
Proteins with quaternary structure
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46. Denaturation
• Denaturation involves protein unfolding, i.e., disruption of higher
orders of protein structure, secondary, tertiary and quaternary
structure (if present), the primary structure remains intact.
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47. Agents that cause denaturation
• The proteins can be denatured by heat,
mechanical pressure or by chemical
denaturants.
• By these agents, the noncovalent
interactions stabilizing the higher orders
of organization are broken, resulting in
unfolding of the polypeptide chain.
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48. Denaturation
• A protein loses its functional
capacity upon denaturation,
showing thereby a close
structure-function relationship.
• Denaturation can be reversible
or irreversible.
• Generally heating or boiling of a
protein results in irreversible
denaturation.
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49. Reversible denaturation
• Ribonuclease is a representative enzyme.
• The functional form of the enzyme is maintained mainly by disulphide
bridges, though other noncovalent interactions also participate in
supporting the tertiary structure.
• In the presence of Urea and β- Mercaptoethanol, the enzyme
undergoes denaturation to form a scrambled structure.
• In that structure, the enzyme loses its overall catalytic capacity.
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50. Protein unfolding
This denaturation is reversible, if the denaturing agents are removed, the disulphide
bridges are reformed, and the enzyme regains its native 3-dimensional functional
form
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51. Molecular chaperones
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52. Protein misfolding
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53. Alzheimer’s disease
Case Details
An 80 –year-old man presented with impairment of brain functions,
alterations of mood, and behavior.
His family reported that he had progressive disorientation and memory
loss over the past six months.
He had trouble handling money and paying bills.
He repeated questions, took longer to complete routine daily tasks, had
poor judgment, and had developed mood and personality changes.
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54. Alzheimer’s disease
There was no family history of dementia.
The routine blood, urine, and C.S.F analysis did not reveal much.
After a computerized tomography (CT) scan and the histopathological
examination of the brain tissue, the patient was diagnosed having
Alzheimer disease.
What is the defect in this disease?
How is the diagnosis made, and what is its prognosis?
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55. Molecular basis of Alzheimer disease
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56. Molecular basis of Alzheimer’s disease
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57. Alzheimer’s disease
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58. Prion disease
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59. Infectious prion disease
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60. Further reading
Please follow the link for details of protein folding diseases
https://www.ourbiochemistry.com/knowledge-base/protein-
misfolding-diseases/
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61. Thank you
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