Alpha Keratin

• Found in hair, fingernails, claws, horns and

beaks
• Sequence consists of 311-314 residue
alpha helical rod segments capped with
non-helical N- and C-termini
• Primary structure of helical rods consists
of 7-residue repeats: (a-b-c-d-e-f-g)n,
where a and d are nonpolar. Promotes
association of helices!

(a) Both type I and type II α-keratin molecules have sequences consisting of long,
central rod domains with terminal cap domains. The numbers of amino acid residues in
each domain are indicated. Asterisks denote domains of variable length. (b) The rod
domains form coiled coils consisting of intertwined right-handed α-helices. These coiled
coils then wind around each other in a left-handed twist. Keratin filaments consist of
twisted protofibrils (each a bundle of four coiled coils). (Adapted from Steinert, P., and Parry,
D., 1985. Annual Review of Cell Biology 1:41–65; and Cohlberg, J., 1993. Trends in Biochemical
Sciences 18:360–362.)

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 Beta Keratin

Proteins that form extensive beta sheets

• Found in silk fibers

• Alternating sequence:
Gly-Ala/Ser-Gly-Ala/Ser....
• Since residues of a beta sheet extend
alternately above and below the plane of the
sheet, this places all glycines on one side and
all alanines and serines on other side!
• This allows Glys on one sheet to mesh with
Glys on an adjacent sheet (same for Ala/Sers)

Silk fibroin consists of a unique stacked array of β-sheets. The primary structure of
fibroin molecules consists of long stretches of alternating glycine and alanine or serine
residues. When the sheets stack, the more bulky alanine and serine residues on one
side of a sheet interdigitate with similar residues on an adjoining sheet. Glycine
hydrogens on the alternating faces interdigitate in a similar manner, but with a smaller
intersheet spacing. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not
to be reproduced without permission.)

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 Collagen - A Triple Helix

Principal component of connective tissue

(tendons, cartilage, bones, teeth)
• basic unit is tropocollagen:
– three intertwined polypeptide chains
(1000 residues each)
– MW = 285,000
– 300 nm long, 1.4 nm diameter
– unique amino acid composition

Collagen

The secrets of its a.a. composition...

• Nearly one residue out of three is Gly
• Proline content is unusually high
• Unusual amino acids found:
– 4-hydroxyproline
– 3-hydroxyproline
– 5-hydroxylysine
– Pro and HyPro together make 30% of
res.

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 Collagen – general structure
• Primary structure
– Repeating Gly-X-Y; X is often Pro
and Y is often hydroxy-Pro
– ~1000 residues
• Secondary structure
– Left-handed helix
• 3 residues/turn
• 0.9 nm/turn
• No intrachain H-bonds
• Tertiary/quaternary structure
– 3 chains form extended right-
handed supercoil

Hydroxylation of proline residues is catalyzed by prolyl hydroxylase. The reaction

requires a -ketoglutarate and ascorbic acid (vitamin C).

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 The Collagen Triple Helix
A case of structure following composition

• The unusual amino acid composition of

collagen is unsuited for alpha helices OR
beta sheets
• But it is ideally suited for the collagen triple
helix: three intertwined helical strands
• Much more extended than alpha helix, with
a rise per residue of 2.9 Angstroms
• 3.3 residues per turn
• Long stretches of Gly-Pro-Pro/HyP

Poly(Gly-Pro-Pro), a collagen-like right-handed triple

helix composed of three left-handed helical chains.
(Adapted from Miller, M. H., and Scheraga, H. A.,
1976, Calculation of the structures of collagen models.
Role of interchain interactions in determining the triple-
helical coiled-coil conformation. I. Poly(glycyl-prolyl-
prolyl). Journal of Polymer Science Symposium
54:171–200.)

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 Structural basis of the collagen
triple helix

• Every third residue faces the crowded center of

the helix - only Gly fits here
• Pro and HyP suit the constraints of phi and psi
• Interchain H-bonds involving HyP stabilize helix
• Fibrils are further strengthened by intrachain
lysine-lysine and interchain hydroxypyridinium
crosslinks

Collagen fibers are

stabilized and
strengthened by Lys – Lys
cross-links. Aldehyde
moieties formed by lysyl
oxidase react in a
spontaneous
nonenzymatic aldol
reaction.

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The hydroxypyridinium structure
formed by the cross-linking of a Lys
and two hydroxy Lys residues.

Hemoglobin
A classic example of allostery
• Hemoglobin and myoglobin are oxygen
transport and storage proteins
• Compare the oxygen binding curves for
hemoglobin and myoglobin
• Myoglobin is monomeric; hemoglobin is
tetrameric
• Mb: 153 aa, 17,200 MW
• Hb: two alphas of 141 residues, 2 betas of
146

7
O2-binding curves for hemoglobin and myoglobin.

The myoglobin and

hemoglobin molecules.
Myoglobin (sperm whale):
one polypeptide chain of 153
amino acid residues (mass =
17.2 kD) has one heme
(mass = 652 D) and binds
one O2. Hemoglobin
(human): four polypeptide
chains, two of 141 amino
acid residues
(α) and two of 146 residues
(β); mass = 64.45 kD. Each
polypeptide has a heme; the
Hb tetramer binds four O2.
(Illustration: Irving Geis Rights
owned by Howard Hughes
Medical Institute. Not to be
reproduced without permission)

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 Detailed structure of the myoglobin
molecule. The myoglobin polypeptide
chain consists of eight helical
segments, designated by the letters A
through H, counting from the N-
terminus. These helices, ranging in
length from 7 to 26 residues, are linked
by short, unordered regions that are
named for the helices they connect, as
in the AB region or the EF region. The
individual amino acids in the
polypeptide are indicated according to
their position within the various
segments, as in His F8, the eighth
residue in helix F, or Phe CD1, the first
amino acid in the interhelical CD
region. Occasionally, amino acids are
specified in the conventional way, that
is, by the relative position in the chain,
as in Gly153. The heme group is
cradled within the folded polypeptide
chain. (Illustration: Irving Geis Rights owned
by Howard Hughes Medical Institute. Not to
be reproduced without permission)

Hemoglobin Function
Hb must bind oxygen in lungs and
release it in capillaries
• When a first oxygen binds to Fe in heme of Hb, the
heme Fe is drawn into the plane of the porphyrin
ring
• This initiates a series of conformational changes that
are transmitted to adjacent subunits
• Adjacent subunits' affinity for oxygen increases
• This is called positive cooperativity

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 Myoglobin Structure
Mb is a monomeric heme protein
• Mb polypeptide "cradles" the heme group
• Fe in Mb is Fe2+ - ferrous iron - the form that
binds oxygen
• Oxidation of Fe yields 3+ charge - ferric iron -
metmyoglobin does not bind oxygen
• Oxygen binds as the sixth ligand to Fe

The six liganding

positions of an iron ion.
Four ligands lie in the
same plane; the
remaining two are,
respectively, above and
below this plane. In
myoglobin, His F8 is the
fifth ligand; in
oxymyoglobin, O2
becomes the sixth.

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Oxygen and carbon monoxide binding to
the heme group of myoglobin.

The Conformation Change

The secret of Mb and Hb!
• Oxygen binding changes the Mb conformation
• Without oxygen bound, Fe is out of heme plane
• Oxygen binding pulls the Fe into the heme plane
• Fe pulls its His F8 ligand along with it
• The F helix moves when oxygen binds
• Total movement of Fe is 0.029 nm - 0.29 A
• This change means little to Mb, but lots to Hb!

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 The displacement of the
Fe ion of the heme of
deoxymyoglobin from
the plane of the
porphyrin ring system by
the pull of His F8. In
oxymyoglobin, the
bound O2 counteracts
this effect.

Binding of Oxygen by Hb
The Physiological Significance
• Hb must be able to bind oxygen in the lungs
• Hb must be able to release oxygen in
capillaries
• If Hb behaved like Mb, very little oxygen
would be released in capillaries
• The sigmoid, cooperative oxygen binding
curve of Hb makes this possible!

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Subunit motion in hemoglobin when the molecule goes from
the (a) deoxy to the (b) oxy form. (Illustration: Irving Geis Rights
owned by Howard Hughes Medical Institute. Not to be reproduced
without permission)

Changes in the
position of the heme
iron atom upon
oxygenation lead to
conformational
changes in the
hemoglobin
molecule.

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Salt bridges between different subunits in hemoglobin. These noncovalent, electrostatic
interactions are disrupted upon oxygenation. Argα141 and Hisβ146 are the C-termini of the
α- and β-polypeptide chains. (a) The various intrachain and interchain salt links formed
among the α - and β-chains of deoxyhemoglobin. (b) A focus on those salt bridges and
hydrogen bonds involving interactions between N-terminal and C-terminal residues in the α-
chains. Note the Cl- ion, which bridges ionic interactions between the N-terminus of α2 and
the R group of Argα141. (c) A focus on the salt bridges and hydrogen bonds in which the
residues located at the C-termini of β-chains are involved. All of these links are abolished in
the deoxy to oxy transition. (Illustration: Irving Geis Rights owned by Howard Hughes Medical Institute.
Not to be reproduced without permission)

The Bohr Effect

Competition between oxygen and H+
• Discovered by Christian Bohr
• Binding of protons diminishes oxygen binding
• Binding of oxygen diminishes proton binding
• Important physiological significance

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 Bohr Effect II
Carbon dioxide diminishes oxygen binding
• Hydration of CO2 in tissues and extremities
leads to proton production
• These protons are taken up by Hb as oxygen
dissociates
• The reverse occurs in the lungs

2,3-Bisphosphoglycerate
An Allosteric Effector of Hemoglobin
• In the absence of 2,3-BPG, oxygen
binding to Hb follows a rectangular
hyperbola!
• The sigmoid binding curve is only
observed in the presence of 2,3-BPG
• Since 2,3-BPG binds at a site distant from
the Fe where oxygen binds, it is called an
allosteric effector

15
The structure, in ionic form, of BPG or
2,3 -bisphosphoglycerate, an important
allosteric effector for hemoglobin.

2,3-BPG and Hb
The "inside" story......
• Where does 2,3-BPG bind?
– "Inside"
– in the central cavity
• What is special about 2,3-BPG?
– Negative charges interact with 2 Lys, 4 His,
2 N-termini
• Fetal Hb - lower affinity for 2,3-BPG, higher
affinity for oxygen, so it can get oxygen from
mother

16
 The ionic binding
of BPG to the two
β-subunits of Hb.
(Illustration: Irving
Geis Rights owned
by Howard Hughes
Medical Institute.
Not to be
reproduced without
permission)

Comparison of the oxygen saturation curves of Hb A and Hb F under similar conditions of

pH and [BPG].

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The polymerization of Hb S via the interactions between the hydrophobic Val side chains at
position β6 and the hydrophobic pockets in the EF corners of β-chains in neighboring Hb
molecules. The protruding “block” on Oxy S represents the Val hydrophobic protrusion.
The complementary hydrophobic pocket in the EF corner of the β-chains is represented by
a square-shaped indentation. (This indentation is probably present in Hb A also.) Only the
β2 Val protrusions and the β1 EF pockets are shown. (The β1 Val protrusions and the β2 EF
pockets are not involved, although they are present.)

Biomedical Implications
• Myoglobinuria – After massive crush
injury, Mb released from muscle fibers
colors the urine dark red.
• Mb can be detected in plasma ff a
myocardial infarction, but assay of serum
enzymes provides a more sensitive index
of myocardial injury.

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 Biomedical Implications
• Anemias – reduction in the # of RBCs or
Hb in the blood, can reflect impaired
synthesis of Hb (in iron deficiency) or
impaired production of erythrocytes (in
folic acid or Vit B12 deficiency). Diagnosis
begins with spectroscopic measurement of
blood Hb levels.

Biomedical Implications
• Thalassemias – genetic defect resulting
from the partial or total absence of one or
more alpha or beta chains of Hb.
• Glycosylated Hb (HbA1c) – reliable means
of monitoring blood glucose level

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 What is an antibody?

• An immunoglobulin
that is capable of
combining with
specificity to the
antigen that elicited its
production.

Schematic drawing of an
immunoglobulin molecule
showing the intramolecular and
intermolecular disulfide bridges.
(A space-filling model of the
same molecule is shown in
Figure 1.11.)

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 What is an antigen?

• An antigen is any substance that elicits an

immune response and is then capable of
binding to the subsequently produced
antibodies.
• Antigens are generally proteins or
polysaccharides, but other substances such
as nucleic acids can also be antigens.

What is an Epitope
• An epitope is the small site on the antigen
which is recognized by the antibody.
• Usually between one and six sugars or
amino acids on the surface of the antigen.
Antibody Uniqueness:
• B-cells produce somewhere between 1 x
108
and 1 x 1010 IgG antibodies with different
binding sites.

21
Schematic drawing of an
immunoglobulin molecule
showing the intramolecular and
intermolecular disulfide bridges.
(A space-filling model of the
same molecule is shown in
Figure 1.11.)

22
Enzyme-Linked Immunosorbent Assay (ELISA)

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