Proteins overview by Vijay Avin BR

1. Protein Structure
and Function
Vijay Avin BR, Molecular Biomedicine Laboratory, Sahyadri
Sceince College, Shimoga, Karnataka, India

2. Our life is maintained byOur life is maintained by
molecular network systemsmolecular network systems
Molecular network
system in a cell
(From ExPASy Biochemical Pathways; http://www.expasy.org/cgi-bin/show_thumbnails.pl?2)

3. Proteins
Make up about 15% of the cell
Have many functions in the cell
Enzymes
Structural
Transport
Motor
Storage
Signaling
Receptors
Gene regulation
Special functions

4. Animals have much more proteins than
in plants (Seeds) in which cellulose
predominates.
Among animals mammals constitutes
largely proteins (Skin, hair, nails,
muscles etc)
Antibodies, enzymes, some harmones
(insulin) are protenatious in nature
Some important facts about proteins

5. It is very important to note htat the tissue
proteins of any two of the individuals are not
identical, except for two twins.
Due to this characteristic, proteins help in
protecting the body by the attack of foreign
toxic proteins and viruses.
The biological importance of proteins can be
judged by the fact that the animals can live
for a long time without fat or carbohydrate,
but not without proteins
Some important facts about proteins

6. Proteins mainly supply new tissue, repair
working parts and make up the loss (eg as
gland secretion) in the vital process.
Only the plants can build up proteins from
inorganic materials, like nitrates, ammonium
sulphate, carbon di oxide and water
While most of the animals derive them mainly
from plants and some other animals
Some important facts about proteins

7. Characteristics of proteins
Most of the proteins are hydrophilic and
some are hydrophobic, high polymer
colloids, few such as insulin, TMV
protein etc are crystalline.
All proteins are leavorotatory, this
property is due to the presence of
alpha-amino acids.
Proteins doesn’t bear any color except
chromoproteins (heamoglobin and
myoglobin)

8. They have no melting point or decomposition
temperature
A pure protein is tasteless and odourless.
Denaturation: on heating, exposing to
ultraviolate radiation or treating with number
of solvents or reagents (alcohol, acetone,
aqueous potassium iodide) the proteins are
precipitated out and thuis undergo
remarkable changes in hteir solubility, optical
rotation and biological properties (eg,
Enzymes become inactive when denatures.
These changes may be irreversible.
Characteristics of proteins

9. Classification of Proteins
Proteins are generally classified on the
basis of increasing complexity in their
structures
1. Simple: which yield only alpha-amino
acids on hydrolysis
Albumin (soluble in water),
globulin(insoluble in water), histamines
etc

10. 2. Conjugated proteins: the conjugated
proteins contain simple protein along
with a non protein group
Glycoproteins, phosphoprteins,
chromoprotein etc
3. Derived proteins: Derived proteins are
the products formed by the action of
physical, chemical or enzymatic agents
on natural proteins
Fibrous and globular proteins

11. Amino acid(s)
mg per kg body
weight
mg per
70 kg
mg per 100 kg Main food sources
H Histidine 10 700 1000
soy protein, eggs, parmesan, sesame,
peanuts[7]
I Isoleucine 20 1400 2000
eggs, soy protein & tofu, whitefish, pork
, parmesan[8]
L Leucine 39 2730 3900
eggs, soy protein, whitefish, parmesan
, sesame[9]
K Lysine 30 2100 3000
eggs, soy protein, whitefish, parmesan
, smelts[10]
M Methionine+ C Cysteine
10.4 + 4.1 (15
total)
1050 1500
eggs, whitefish, sesame, smelts,
soy protein[11]
+ eggs, soy protein,
sesame, mustard seeds,peanuts[12]
FPhenylalanine+ Y Tyrosine 25 (total) 1750 2500
eggs, soy protein, peanuts, sesame,
whitefish[13]
+ soy protein, eggs,
parmesan, sesame[14]
T Threonine 15 1050 1500
eggs, soy protein, whitefish, smelts,
sesame[15]
W Tryptophan 4 280 400
soy protein, sesame, eggs,
winged beans, chia seeds[16]
V Valine 26 1820 2600
eggs, soy protein, parmesan, sesame,
beef[17]

12. 12
Fibrous proteins have a structural
role
•Collagen is the most abundant protein inCollagen is the most abundant protein in
vertebrates. Collagen fibers are a majorvertebrates. Collagen fibers are a major
portion of tendons, bone and skin. Alphaportion of tendons, bone and skin. Alpha
helices of collagen make up a triple helixhelices of collagen make up a triple helix
structure giving it tough and flexiblestructure giving it tough and flexible
properties.properties.
•Fibroin fibers make the silk spun by spidersFibroin fibers make the silk spun by spiders
and silk worms stronger weight for weightand silk worms stronger weight for weight
than steel! The soft and flexible propertiesthan steel! The soft and flexible properties
come from the beta structure.come from the beta structure.
•Keratin is a tough insoluble protein thatKeratin is a tough insoluble protein that
makes up the quills of echidna, your hair andmakes up the quills of echidna, your hair and
nails and the rattle of a rattle snake. Thenails and the rattle of a rattle snake. The
structure comes from alpha helices that arestructure comes from alpha helices that are
cross-linked by disulfide bonds.cross-linked by disulfide bonds.

13. 13
The globular proteins
The globular proteins have a number of biologically important roles. They
include:
Cell motility – proteins link together to form filaments which make
movement possible.
Organic catalysts in biochemical reactions – enzymes
Regulatory proteins – hormones, transcription factors
Membrane proteins – MHC markers, protein channels, gap junctions
Defense against pathogens – poisons/toxins, antibodies, complement
Transport and storage – hemoglobin and myosin

14. 14
Proteins for cell motility
Myosin (red) and actin filaments (green)
in coordinated muscle contraction.
Actin bound to the mysoin binding site
(groove in red part of myosin protein).
Add energy (ATP) and myosin moves,
moving actin with it.
The sperm motility is activated by changes in
intracellular ion concentration. The change in
concentration that signals the mechanism is different
among species. In marine invertebrates and
sea urchins, the rise in pH to about 7.2-7.6 activates
ATPase which leads to decrease in potassium, thus
induces membrane hyperpolarization. As a result,
sperm motility is activated. The change in cell volume
which alters intracellular ion concentration can also
contribute to the activation of sperm motility. In
some mammals, sperm motility is activated by
increase in pH, calcium ion and cAMP,

15. 15
Eukaryote cells have a cytoskeleton made up of straight
hollow cylinders called microtubules (bottom left).
They help cells maintain their shape, they act like conveyer
belts moving organelles around in the cytoplasm, and they
participate in forming spindle fibres in cell division.
Microtubules are composed of filaments of the protein,
tubulin (top left) . These filaments are compressed like
springs allowing microtubules to ‘stretch and contract’.
13 of these filaments attach side to side, a little like the
slats in a barrel, to form a microtubule. This barrel shaped
structure gives strength to the microtubule.
Tubulin
forms
helical
filaments
Proteins in the Cell Cytoskeleton

16. 16
Catalase speeds up the
breakdown of
hydrogen peroxide,
(H2O2) a toxic by
product of metabolic
reactions, to the
harmless substances,
water and oxygen.
The reaction is extremely
rapid as the enzyme
lowers the energy
needed to kick-start
the reaction (activation
energy)
Energy
Progress of reaction
Substrate Product
No catalyst =No catalyst =
Input of 71kJ energy requiredInput of 71kJ energy required
Activation
Energy
With catalaseWith catalase
= Input of 8 kJ energy required= Input of 8 kJ energy required
Proteins speed up reactions - EnzymesProteins speed up reactions - Enzymes
+2 2

17. 17
Proteins can regulate metabolism –
hormones
When your body detects an increase in the sugar
content of blood after a meal, the hormone
insulin is released from cells in the pancreas.
Insulin binds to cell membranes and this triggers the
cells to absorb glucose for use or for storage as
glycogen in the liver.
Proteins span membranes –protein channelsProteins span membranes –protein channels
Source: http://www.biology.arizona.edu/biochemistry/tutorials/chemistry/page2.html
http://www.cbp.pitt.edu/bradbury/projects.htm
The CFTR membrane protein is an ion channel that
regulates the flow of chloride ions.
Not enough of this protein gets inserted into the
membranes of people suffering Cystic fibrosis. This causes
secretions to become thick as they are not hydrated. The
lungs and secretory ducts become blocked as a
consequence.

18. 18
Proteins Defend us against pathogens –
antibodies
Left: Antibodies like IgG found in
humans, recognise and bind to
groups of molecules or epitopes
found on foreign invaders.
Right: The binding site of an antigen
protein (left) interacting with the
epitope of a foreign antigen (green)

19. Protein structure
Protein structure is the biomolecular structure of a protein molecule.
Each protein is a polymer – specifically a polypeptide – that is a
sequence formed from various L-α-amino acids (also referred to as
residues).
By convention, a chain under 40 residues is often identified as a
peptide, rather than a protein. To be able to perform their biological
function, proteins fold into one or more specific spatial conformations,
driven by a number of non-covalent interactions such as
hydrogen bonding, ionic interactions, Van der Waals forces, and
hydrophobic packing.
To understand the functions of proteins at a molecular level, it is often
necessary to determine their three-dimensional structure. This is the
topic of the scientific field of structural biology, which employs
techniques such as X-ray crystallography, NMR spectroscopy, and
dual polarisation interferometry to determine the structure of proteins.

20. Amino acid: Basic unit ofAmino acid: Basic unit of
proteinprotein
COO-
NH3
+
C
R
H
An amino
acid
Different side chains,
R, determin the
properties of 20
amino acids.
Amino group Carboxylic
acid group

21. 20 Amino acids20 Amino acids
Glycine (G)
Glutamic acid (E)Asparatic acid (D)
Methionine (M)
Threonine (T)Serine (S)Glutamine (Q)
Asparagine (N)Tryptophan (W)Phenylalanine (F)
Cysteine (C)
Proline (P)
Leucine (L)Isoleucine (I)Valine (V)Alanine (A)
Histidine (H)Lysine (K)
Tyrosine (Y)
Arginine (R)
White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic

22. Proteins are linear polymers ofProteins are linear polymers of
amino acidsamino acids
R1
NH3
＋
C CO
H
R2
NH C CO
H
R3
NH C CO
H
R2
NH3
＋
C CO
O ー
H
＋
R1
NH3
＋
C CO
O ー
H
＋
H2OH2O
Peptide
bond
Peptide
bond
The amino acid
sequence is called as
primary structure
A A
F
NG
G
S
T
S
D
K
A carboxylic acid
condenses with an amino
group with the release of a
water

23. Amino acid sequence isAmino acid sequence is
encoded by DNA base sequenceencoded by DNA base sequence
in a genein a gene
・
C
G
C
G
A
A
T
T
C
G
C
G
・
・
G
C
G
C
T
T
A
A
G
C
G
C
・
DNA
molecule
＝
DNA base
sequence

24. Amino acid sequence isAmino acid sequence is
encoded by DNA base sequenceencoded by DNA base sequence
in a genein a gene
Second letter
T C A G
Firstletter
T
TTT Phe TCT
Ser
TAT Tyr TGT Cys T
Thirdletter
TTC TCC TAC TGC C
TTA Leu TCA TAA Stop TGA Stop A
TTG TCG TAG TGG Trp G
C
CTT
Leu
CCT
Pro
CAT His CGT
Arg
T
CTC CCC CAC CGC C
CTA CCA CAA
Gln
CGA A
CTG CCG CAG CGG G
A
ATT
Ile
ACT
Thr
AAT Asn AGT Ser T
ATC ACC AAC AGC C
ATA ACA AAA Lys AGA Arg A
ATG Met ACG AAG AGG G
G
GTT
Val
GCT
Ala
GAT
Asp
GGT
Gly
T
GTC GCC GAC GGC C
GTA GCA GAA Glu GGA A
GTG GCG GAG GGG G

25. Gene is protein’s blueprint,Gene is protein’s blueprint,
genome is life’s blueprintgenome is life’s blueprint
Gene
GenomeDNA
Protein
Gene Gene
Gene
Gene
Gene
Gene
GeneGene
GeneGene
GeneGene
Gene
Gene
Protein Protein
Protein
Protein
Protein
ProteinProtein
Protein
Protein
Protein
Protein
Protein
Protein
Protein

26. Gene is protein’s blueprint,Gene is protein’s blueprint,
genome is life’s blueprintgenome is life’s blueprint
Genome
Gene Gene
Gene
Gene
Gene
Gene
GeneGene
GeneGene
GeneGene
Gene
Gene
Protein Protein
Protein
Protein
Protein
ProteinProtein
Protein
Protein
Protein
Protein
Protein
Protein
Protein
Glycolysis network

27. 3 billion base pair => 6 G letters
&
1 letter => 1 byte
The whole genome can be
recorded in just 10 CD-ROMs!
In 2003, Human genomeIn 2003, Human genome
sequence was deciphered!sequence was deciphered!
Genome is the complete set of genes of a living thing.
In 2003, the human genome sequencing was completed.
The human genome contains about 3 billion base pairs.
The number of genes is estimated to be between 20,000
to 25,000.
The difference between the genome of human and that of
chimpanzee is only 1.23%!

28. Hierarchical nature of proteinHierarchical nature of protein
structurestructure
Primary structure (Amino acid sequence)
↓
Secondary structure （ α-helix, β-sheet ）
↓
Tertiary structure （ Three-dimensional structure
formed by assembly of secondary structures ）
↓
Quaternary structure （ Structure formed by more
than one polypeptide chains ）

29. Basic structural units of proteins:Basic structural units of proteins:
Secondary structureSecondary structure
α-helix β-sheet
Secondary structures, α-helix
and β-sheet, have regular
hydrogen-bonding patterns.

30. The primary structure refers to amino acid linear sequence of the
polypeptide chain. The primary structure is held together by
covalent bonds such as peptide bonds, which are made during the
process of protein biosynthesis or translation. The two ends of the
polypeptide chain are referred to as the carboxyl terminus (C-terminus)
and the amino terminus (N-terminus) based on the nature of the free
group on each extremity. Counting of residues always starts at the N-
terminal end (NH2-group), which is the end where the amino group is not
involved in a peptide bond.
Primary structure
Post-translational modifications such as disulfide
formation, phosphorylations and glycosylations
are usually also considered a part of the primary
structure, and cannot be read from the gene.
Example: Insulin is composed of 51 amino acids
in 2 chains. One chain has 31 amino acids and the
other has 20 amino acids.

32. Secondary structure refers to highly regular local sub-structures. Two main
types of secondary structure, the alpha helix and the beta strand or
beta sheets, were suggested in 1951 by Linus Pauling and coworkers.
These secondary structures are defined by patterns of hydrogen bonds
between the main-chain peptide groups. They have a regular geometry, being
constrained to specific values of the dihedral angles ψ and φ on the
Ramachandran plot.
Both the alpha helix and the beta-sheet represent a way of saturating all the
hydrogen bond donors and acceptors in the peptide backbone. Some parts of
the protein are ordered but do not form any regular structures. They should not
be confused with random coil, an unfolded polypeptide chain lacking any fixed
three-dimensional structure. Several sequential secondary structures may form
a "super secondary unit"
Secondary structure

33. 33
Alpha Helix
A helix can turn right
or left from N to C
terminus – only
right-handed are
observed in nature
as this produces
less clashes
All hydrogen bonds
are satisfied except
at the ends = stable

34. 34
Alpha Helix Continued
There are 3.6
residues per turn
A helical wheel will
outline the surface
properties of the
helix

35. 35
Other (Rarer) Helix Types - 310
Less favorable
geometry
3 residues per turn
with i+3 not i+4
Hence narrower and
more elongated
Usually seen at the
end of an alpha
helix

36. 36
Other (Very Rare) Helix Types -
Π
Less favorable geometry
4 residues per turn with i+5 not i+4
Squat and constrained

37. 37
Beta Sheets

38. 38
Beta Sheets Continued
Between adjacent polypeptide chains
Phi and psi are rotated approximately 180 degrees
from each other
Mixed sheets are less common
Viewed end on the sheet has a right handed twist
that may fold back upon itself leading to a barrel
shape (a beta barrel)
Beta bulge is a variant; residue on one strand forms
two hydrogen bonds with residue on other – causes
one strand to bulge – occurs most frequently in
parallel sheets

39. 39
Other Secondary Structures –
Loop or Coil
Often functionally significant
Different types
Hairpin loops (reverse turns) – often
between anti-parallel beta strands
Omega loops – beginning and end close
(6-16 residues)
Extended loops – more than 16 residues

40. Tertiary structure refers to three-dimensional structure of a single
protein molecule. The alpha-helices and beta-sheets are folded into a
compact globule. The folding is driven by the non-specific
hydrophobic interactions (the burial of hydrophobic residues from
water), but the structure is stable only when the parts of a protein
domain are locked into place by specific tertiary interactions, such as
salt bridges, hydrogen bonds, and the tight packing of side chains
anddisulfide bonds. The disulfide bonds are extremely rare in
cytosolic proteins, since the cytosol is generally a reducing
environment.
Tertiary structure

41. Protein are frequently described as consisting from several structural units.
A structural domain is an element of the protein's overall structure that is
self-stabilizing and often folds independently of the rest of the protein chain.
Many domains are not unique to the protein products of one gene or one
gene family but instead appear in a variety of proteins. Domains often are
named and singled out because they figure prominently in the biological
function of the protein they belong to; for example, the "calcium-binding
domain of calmodulin". Because they are independently stable, domains can
be "swapped" by genetic engineering between one protein and another to
make chimeras.
The structural and sequence motifs refer to short segments of protein
three-dimensional structure or amino acid sequence that were found in a
large number of different proteins.
The super secondary structure refers to a specific combination of
secondary structure elements, such as beta-alpha-beta units or
helix-turn-helix motif. Some of them may be also referred to as structural
motifs.
Protein fold refers to the general protein architecture, like helix bundle,
Domains, motifs, and folds in protein structure

42. 42
Tertiary Structure as Dictated by
the Environment
Proteins exist in an aqueous environment where hydrophilic
residues tend to group at the surface and hydrophobic residues
form the core – but the backbone of all residues is somewhat
hydrophilic – therefore it is important to have this neutralized by
satisfying all hydrogen bonds as is achieved in the formation of
secondary structures
Polar residues must be satisfied in the same way – on occasion
pockets of water (discreet from the solvent) exist as an intrinsic
part of the protein to satisfy this need
Ion pairs (aka salt bridge) form important interactions
Disulphide linkages between cysteines form the strongest (ie
covalent tertiary linkages); the majority of cysteines do not form
such linkages

43. 43
Tertiary Structure as Dictated by
Protein Modification
To the amino acid itself
eg hydroxyproline
needed for collagen
formation
Addition of
carbohydrates
(intracellular
localization)
Addition of lipids
(binding to the
membrane)
Association with small
molecules – notably
metals eg hemoglobin

44. 44
There are Different Forms of
Classification apart from
Structural
Biochemical
Globular
Membrane
Fibrous
myoglobin
Collagen
Bacteriorhodopsin

45. Quaternary structure is the three-dimensional structure of a multi-subunit
protein and how the subunits fit together. In this context, the quaternary
structure is stabilized by the same non-covalent interactions and
disulfide bonds as the tertiary structure. Complexes of two or more
polypeptides (i.e. multiple subunits) are called multimers.
Specifically it would be called a dimer if it contains two subunits, a trimer
if it contains three subunits, and a tetramer if it contains four subunits.
The subunits are frequently related to one another by
symmetry operations, such as a 2-fold axis in a dimer. Multimers made
up of identical subunits are referred to with a prefix of "homo-" (e.g. a
homotetramer) and those made up of different subunits are referred to
with a prefix of "hetero-" (e.g. a heterotetramer, such as the two alpha
and two beta chains of hemoglobin).
Quaternary structure

46. Three-dimensional structure ofThree-dimensional structure of
proteinsproteins
Tertiary
structure
Quaternary structure

47. Keratin
Keratin is a family of
fibrous structural proteins. Keratin is
the key structural material making
up the outer layer of human skin.
In general Keratin is the protein that
protects the epithelial cells from
damage and stress that could kill
the cell.
It is also the key structural
component of hair and nails.
Keratin monomers assemble into
bundles to form
intermediate filaments, which are
tough and insoluble and form
strong unmineralized tissues found
inreptiles, birds, amphibians, and
mammals.

48. The average molecular weight of
Keratin-7 is 54kD
· In one case, the human molecular
weight of Keratin 7 was 51.4 kD.
The Chromosome Location of Keratin
This means that it is located on the 12th
human chromosome.

49. alpha (cysteine rich) isomer found in
cytoskeleton and hair.
beta (cysteine poor) isomer found mostly in birds
and reptiles. It is the building block of scales,
feathers and claws. It is rich in residues with
small side chains: glycine, alanine and serine.
alpha form can be stretched up to 120% in moist
heat. beta form is rigid.
Cysteine can form disulfide bridges with other
cysteine residues. These cross-linkages
decrease the elasticity of alpha-keratin.

50. Keratin-Etymology
the α-keratins in the hair (including
wool), horns, nails, claws and hooves
of mammals.
the harder β-keratins found in nails and
in the scales and claws of reptiles,
their shells (Testudines, such as
tortoise, turtle, terrapin), and in the
feathers, beaks, claws of birds and
quills of porcupines. (These keratins
are formed primarily in beta sheets.
However, beta sheets are also found in
α-keratins.)
The baleen plates of filter-feeding
whales are made of keratin.

52. In the early 1950s Linus Pauling and R.B. Corey
in proposed several structures for keratin.
Observed shorter than expected amide C-N
bond. They deduced that the peptide bond
was planar.
A planar peptide bond reduced the number of
conformations of a poly-peptide chain and led
to their proposal of the alpha helix and the
beta sheet.
alpha-helix explained the x-ray data which
showed a repeat unit of 0.50 – 0.55 nm. This
distance corresponds to the height of the rise
per revolution of helix.
alpha-helix also explained a repeat unit of
0.15 nm. This distance corresponds to the
height of the rise per residue. The ratio of
these two numbers give the number of amino
acids per revolution: 3.6
Hydrogen bonding occurs between carbonyl
oxygen and the amide hydrogen on next twist
of helix.

53. In a coil group of 7 residues, 1st & 4th positions contain
hydrophobic aa’s
These nonpolar aa’s on different helical chains attract each
other and make up the inside positions of the double coils
These hydrophobic reactions stabilize the coil structure
The outside positions are mostly polar aa’s

54. Fibroin
Fibroin is an insoluble protein created by spiders,
the larvae of Bombyx mori, other moth genera such
as Antheraea, Cricula, Samia and Gonometa, and
numerous other insects.
Silk in its raw state consists of two main proteins,
sericin and fibroin, fibroin being the structural center
of the silk, and sericin being the sticky material
surrounding it.

55. Hemoglobin and Myoglobin
Because of its red color, the red blood pigment has been of
interest since antiquity.
•First protein to be crystallized - 1849.
•First protein to have its mass accurately measured.
•First protein to be studied by ultracentrifugation.
•First protein to associated with a physiological
condition.
•First protein to show that a point mutation can cause
problems.
•First proteins to have X-ray structures determined.
•Theories of cooperativity and control explain
hemoglobin function

56. The structure of myoglobin
Andrew Kendrew and Max Perutz solved the structure of these
molecules in 1959 to 1968.
Myoglobin: 44 x 44 x 25 Å single subunit 153 amino acid
residues
121 residues are in an a helix. Helices are named A, B, C, …F.
The heme pocket is surrounded by E and F but not B, C, G, also
H is near the heme.
Amino acids are identified by the helix and position in the helix
or by the absolute numbering of the residue.
shared the 1962 Nobel Prize in chemistry with

57. Myoglobin is the primary oxygen-carrying pigment of muscle tissues. High
concentrations of myoglobin in muscle cells allow organisms to hold their
breaths longer. Diving mammals such as whales and seals have muscles with
particularly high myoglobin abundance
Myoglobin forms pigments responsible for making meat red. The color that meat takes
is partly determined by the oxidation states of the iron atom in myoglobin and the
oxygen species attached to it. When meat is in its raw state, the iron atom is in the +2
oxidation state, and is bound to a dioxygen molecule (O2). Meat cooked well done is
brown because the iron atom is now in the +3 oxidation state, having lost an electron,
and is now coordinated by a water molecule. Under some conditions, meat can also
remain pink all through cooking, despite being heated to high temperatures. If meat has
been exposed to nitrites, it will remain pink because the iron atom is bound to
NO, nitric oxide (true of, e.g., corned beef or cured hams).

58. The Backbone structure of Myoglobin 58
Heme
prosthetic group

59. Heme Prosthetic Group
Heme (Fe2+
) has
affinity for O2.
Hematin (Fe3+
)
cannot bind O2.
Located in crevice
where it is protected
from oxidation.
N
N N
N
HO
O
Fe

60. Oxygen Binding to Myoglobin
O2 binds to only
available coordination
site on iron atom.
His 93 (proximal his)
binds directly to iron.
His 64 (distal his)
stabilizes the O2 binding
site.
http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG04_44.JPG
distal histidine
proximal histidine

61. Hemoglobin
Spherical 64 x 55 x 50 Å two fold rotation of
symmetry α and β subunits are similar and are placed
on the vertices of a tetrahedron. There is no D helix in
the α chain of hemoglobin. Extensive interactions
between unlike subunits α2-β2 or α1-β1 interface
has 35 residues while α1-β2 and α2-β1 have 19
residue contact.
Oxygenation causes a considerable structural
conformational change

62. O2 Binding and Allosteric
Properties of Hemoglobin
• Hemoglobin binds and transports HHemoglobin binds and transports H++
, O, O22 andand
COCO22 in an allosteric mannerin an allosteric manner
• Allosteric interaction –Allosteric interaction – of, relating to, undergoing, or being aof, relating to, undergoing, or being a
change in the shape and activity of a protein (as an enzyme) that resultschange in the shape and activity of a protein (as an enzyme) that results
from combination with another substance at a point other than thefrom combination with another substance at a point other than the
chemically active sitechemically active site
• a regulatory mechanism where a smalla regulatory mechanism where a small
molecule (effector) binds and alters anmolecule (effector) binds and alters an
enzymes activityenzymes activity

63. • There are two general structural states - the deoxy or T formThere are two general structural states - the deoxy or T form
and the oxy or R form.and the oxy or R form.
One type of interactions shift is the polar bonds between theOne type of interactions shift is the polar bonds between the
alpha 1 and the beta 2 subunits.alpha 1 and the beta 2 subunits.
The two states

64. The T form finds the terminals in
several important H bonds and
salt bridges.
In the T form the C terminus of
each subunit are "locked" into
position through several
hydrogen and ionic bonds.
Shifts into the R state break
these and allow an increased
movement throughout the
molecule.
Note that binding of one or more
oxygen can have a dramatic affect
on the other subunits that have not
yet bound an O2.

65. Quaternary structure of deoxy- and oxyhemoglobin
T-state R-state

66. Oxygenation rotates the α1β1 dimer in relation to
α2β2 dimer about 15°
The conformation of the deoxy state is called the T state
The conformation of the oxy state is called the R state
individual subunits have a t or r if in the deoxy or oxy state.
What causes the differences in the
conformation states?
It is somehow associated with the binding of
oxygen, but how?

67. The positive cooperativity of O2 binding to Hb
arises from the effect of the ligand-binding
state of one heme on the ligand-binding
affinity of another.
The Fe iron is about 0.6 Å out of the heme
plane in the deoxy state. When oxygen binds
it pulls the iron back into the heme plane.
Since the proximal His F8 is attached to the
Fe this pulls the complete F helix like a lever
on a fulcrum.

69. Binding of the oxygen on one heme is more difficult
but its binding causes a shift in the α1-β2 contacts
and moves the distal His E7 and Val E11 out of the
oxygen’s path to the Fe on the other subunit. This
process increases the affinity of the heme toward
oxygen.
The α1-β2 contacts have two stable positions.
These contacts, which are joined by different but
equivalent sets of hydrogen bonds and act as a
binary switch between the T and the R states

70. The energy in the formation of the Fe-O2 bond
formation drives the T→ R transition.
Hemoglobins O2 -binding Cooperativity derives from
the T → R Conformational shift.
•The Fe of any subunit cannot move into its heme plane
without the reorientation of its proximal His so as to prevent
this residue from bumping into the porphyrin ring.
•The proximal His is so tightly packed by its surrounding
groups that it can not reorient unless this movement is
accompanied by the previously described translation of the F
helix across the heme plane.
•The F helix translation is only possible in concert with the
quaternary shift that steps the α1C-β2FG contact one turn
along the α1C helix.

71. •The inflexibility of the α1-β1 and the α2-β2 interfaces requires
that this shift simultaneously occur at both the α1-β2 and α2-β1
interfaces.
No one subunit or dimer can change its conformation.
The t state with reduced oxygen affinity will be
changed to the r state without binding oxygen
because the other subunits switch upon oxygen
binding. Unbound r state has a much higher affinity
for oxygen, and this is the rational for cooperativity

72. Hemoglobin function
α2,β2 dimer which are structurally similar to myoglobin
•Transports oxygen from lungs to tissues.
•O2 diffusion alone is too poor for transport in larger
animals.
•Solubility of O2 is low in plasma i.e. 10-4
M.
•But bound to hemoglobin, [O2] = 0.01 M or that of air
•Two alternative O2 transporters are;
•Hemocyanin, a Cu containing protein.
•Hemoerythrin , a non-heme containing protein.

73. Function of the globin
Protoporphyrin binds oxygen to the sixth ligand of
Fe(II) out of the plane of the heme. The fifth ligand
is a Histidine, F8 on the side across the heme plane.
His F8 binds to the proximal side and the oxygen
binds to the distal side.
The heme alone interacts with oxygen such that the
Fe(II) becomes oxidized to Fe(III) and no longer
binds oxygen.

74. Fe O O Fe
A heme dimer is formed
which leads to the
formation of Fe(III)
By introducing steric hindrance on one side of the heme plane
interaction can be prevented and oxygen binding can occur.
The globin acts to:
•a. Modulate oxygen binding
affinity
•b. Make reversible oxygen
binding possible

75. The globin surrounds the heme like a hamburger
is surrounded by a bun. Only the propionic acid
side chains are exposed to the solvent.
Amino acid mutations in the heme pocket can
cause autooxidation of hemoglobin to form
methemoglobin.

76. The Bohr Effect
Higher pH i.e. lower [H+
] promotes tighter binding of
oxygen to hemoglobin
and
Lower pH i.e. higher [H+] permits the easier release of
oxygen from hemoglobin
( ) ( ) +
+
+⇔+ xHOHbOHOHb 1n22xn2
Where n = 0, 1, 2, 3 and x ≅ 0.6 A shift in the equilibrium
will influence the amount of oxygen binding. Bohr protons

77. Molecular chaperons

78. Macromolecular crowdingMacromolecular crowding
When doing experiments in vitro, we
should all be thinking about this:
proteins in isolated (pure) systems may not
behave as they do in the cell
- binding partner(s) might be missing
- cell conditions (pH, salts, etc)
- post-translational modifications might be missing
may be dramatically different
This condition is known as molecular
crowding

79. Effects of crowdingEffects of crowding
Definition:
Molecular crowding is a generic term for the condition where a significant
volume of a solution, or cytoplasm for example, is occupied with things other
than water
Fact:
- association constants (ka) increase significantly
- dissociation constants (kd) decrease significantly (kd=1/ka)
- increased on-rates for protein-protein interactions
Assumption:
- non-native polypeptides will have greater tendency to associate
intermolecularly, enhancing the propensity of aggregation

80. Problem:Problem: non-native proteinsnon-native proteins
• non-native proteins expose hydrophobic residues that are
normally buried within the ‘core’ of the protein
• these hydrophobic amino acids have a strong tendency to
interact with other hydrophobic (apolar) residues
- especially under crowding conditions
intramolecular
misfolding
X
X
X
X
intermolecular
aggregation
X
X
X
X
X
X
incorrect
molecular
interactions
&
loss of activity
exposed
hydrophobic
residues

81. Solution:Solution: molecular chaperonesmolecular chaperones
• in the late 1970’s, the term molecular chaperone was coined to
describe the properties of nucleoplasmin:
Nucleoplasmin prevents incorrect interactions between histones and DNA
Dictionary definition:
1: a person (as a matron) who for propriety accompanies one or more young unmarried
women in public or in mixed company
2: an older person who accompanies young people at a social gathering to ensure
proper behavior; broadly : one delegated to ensure proper behavior
• in the late 1980’s, the term molecular chaperone was used more
broadly by John Ellis to describe the roles of various cellular
proteins in protein folding and assembly

82. Molecular chaperones:Molecular chaperones:
general conceptsgeneral concepts
Requirements for a protein to be considered a chaperone:
(1) interacts with and stabilizes non-native forms of protein(s)
- technically also: folded forms that adopt different protein conformations
(2) not part of the final assembly of protein(s)
Functions of a chaperone:
“classical”
- assist folding and assembly
more recent
- modulation of conformation
- transport
- disaggregation of protein aggregates
- unfolding of proteins
assisted
self-assembly
(as opposed to spontaneous
self-assembly)
assisted
disassembly
prevention of assembly
self-assembly refers to the folding of the polypeptide, as well as to
its assembly into functional homo- or hetero-oligomeric structures

83. Molecular chaperones:Molecular chaperones:
common functional assayscommon functional assays
Type of assay Rationale
Binary complex
formation
If chaperone has high enough affinity for an unfolded
polypeptide, it will form a complex detectable by:
• co-migration by SEC;
• co-migration by native gel electrophoresis
• co-immunoprecipitation
Prevention of
aggregation
Binding of chaperones to non-native proteins often
reduces or eliminates their tendency to aggregate. Assay
may detect weaker interactions than is possible with SEC
Refolding
Chaperones stabilize non-native proteins; some can assist
the refolding of the proteins to their native state. Usually,
chaperones that assist refolding are ATP-dependent
Assembly Some chaperones assist protein complex assembly
Activity
Some chaperones modulate the conformation/activity of
proteins
(Miscellaneous) A number of chaperones have specialized functions

84. Human chaperone proteins
Chaperones are found in, for example, the endoplasmic reticulum
(ER), since protein synthesis often occurs in this area.
Endoplasmic reticulum
In the endoplasmic reticulum (ER) there are general, lectin- and
non-classical molecular chaperones helping to fold proteins.
•General chaperones: GRP78/BiP, GRP94, GRP170.
(Binding immunoglobulin protein (BiP) also known as 78 kDa
glucose-regulated protein (GRP-78) or heat shock 70 kDa
protein 5 (HSPA5) is a protein that in humans is encoded by
the HSPA5 gene)
•Lectin chaperones: calnexin and calreticulin
•Non-classical molecular chaperones: HSP47 and ERp29
•Folding chaperones:
-Protein disulfide isomerase (PDI),
-Peptidyl prolyl cis-trans-isomerase (PPI),
-ERp57

85. The secondary structures that polypeptides can adopt in proteins are governed by
hydrogen bonding interactions between the electronegative carbonyl oxygen atoms and
the electropositive amide hydrogen atoms in the backbone chain of the molecule.
These hydrogen-bonding interactions can form the framework that stabilizes the
secondary structure. Many secondary structures with reasonable hydrogen bonding
networks could be proposed but we see only a few possibilities in polypeptides composed
of L-amino acids (proteins). Most of the possible secondary structures are not possible
due to limits on the configuration of the backbone of each amino acid residue.
Understanding these limitations will help to understand the secondary structures of
proteins.
the regions in the set of possible amino acid configurations that are allowed and
disallowed in proteins. This set of values is often graphically represented as a
Ramachandran diagram.

86. ……..End……..