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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 righthanded 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 triplehelical coiled-coil conformation. I. Poly(glycyl-prolylprolyl). 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

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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 Nterminus. 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 αchains. 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

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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

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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.

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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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Enzyme-Linked Immunosorbent Assay (ELISA)

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