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Alpha Keratin

• Found in hair, fingernails, claws, horns andbeaks

• Sequence consists of 311-314 residuealpha helical rod segments capped withnon-helical N- and C-termini

• Primary structure of helical rods consistsof 7-residue repeats: (a-b-c-d-e-f-g)n,where a and d are nonpolar. Promotesassociation 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 ineach domain are indicated. Asterisks denote domains of variable length. (b) The roddomains form coiled coils consisting of intertwined right-handed α -helices. These coiledcoils then wind around each other in a left-handed twist. Keratin filaments consist oftwisted 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 BiochemicalSciences 18:360–362 .)



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 thesheet, this places all glycines on one side andall 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 offibroin molecules consists of long stretches of alternating glycine and alanine or serineresidues. When the sheets stack, the more bulky alanine and serine residues on oneside of a sheet interdigitate with similar residues on an adjoining sheet. Glycinehydrogens on the alternating faces interdigitate in a similar manner, but with a smallerintersheet spacing. (Illustration: Irving Geis. Rights owned by Howard Hughes Medical Institute. Not

to be reproduced without permission.)



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% ofres.



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



A case of structure following composition

• The unusual amino acid composition ofcollagen is unsuited for alpha helices ORbeta sheets

• But it is ideally suited for the collagen triplehelix: three intertwined helical strands

• Much more extended than alpha helix, witha rise per residue of 2.9 Angstroms

• 3.3 residues per turn

• Long stretches of Gly-Pro-Pro/HyP

The Collagen Triple Helix

Poly(Gly-Pro-Pro), a collagen-like right-handed triplehelix 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 Symposium54:171–200 .)



• Every third residue faces the crowded center ofthe 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 intrachainlysine-lysine and interchain hydroxypyridiniumcrosslinks

Structural basis of the collagen triple helix

Collagen fibers arestabilized andstrengthened by Lys – Lyscross-links. Aldehydemoieties formed by lysyloxidase react in aspontaneousnonenzymatic aldol

reaction.



The hydroxypyridinium structureformed by the cross-linking of a Lysand 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



O2-binding curves for hemoglobin and myoglobin.

The myoglobin andhemoglobin molecules.

Myoglobin (sperm whale):one polypeptide chain of 153amino acid residues (mass =17.2 kD) has one heme(mass = 652 D) and bindsone O2. Hemoglobin (human): four polypeptidechains, two of 141 aminoacid residues

(α) and two of 146 residues

(β); mass = 64.45 kD. Eachpolypeptide has a heme; theHb tetramer binds four O2.(Illustration: Irving Geis Rights

owned by Howard Hughes Medical Institute. Not to be reproduced without permission)



Detailed structure of the myoglobinmolecule. The myoglobin polypeptidechain consists of eight helicalsegments, designated by the letters A

through H, counting from the N-terminus. These helices, ranging inlength from 7 to 26 residues, are linkedby short, unordered regions that arenamed for the helices they connect, asin the AB region or the EF region. Theindividual amino acids in thepolypeptide are indicated according totheir position within the varioussegments, as in His F8, the eighth

residue in helix F, or Phe CD1, the firstamino acid in the interhelical CDregion. Occasionally, amino acids arespecified in the conventional way, thatis, by the relative position in the chain,

as in Gly153. The heme group is

cradled within the folded polypeptidechain. (Illustration: Irving Geis Rights owned

by Howard Hughes Medical Institute. Not to be reproduced without permission)

Hemoglobin FunctionHb 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 porphyrinring

• This initiates a series of conformational changes thatare transmitted to adjacent subunits

• Adjacent subunits' affinity for oxygen increases• This is called positive cooperativity



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 ligandingpositions of an iron ion.Four ligands lie in thesame plane; theremaining two are,respectively, above andbelow this plane. Inmyoglobin, His F8 is thefifth ligand; inoxymyoglobin, O2

becomes the sixth.



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!



The displacement of the

Fe ion of the heme ofdeoxymyoglobin fromthe plane of the

porphyrin ring system bythe pull of His F8. In

oxymyoglobin, thebound O2 counteractsthis 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!



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 theposition of the hemeiron atom uponoxygenation lead toconformationalchanges in thehemoglobinmolecule.



Salt bridges between different subunits in hemoglobin. These noncovalent, electrostatic

interactions are disrupted upon oxygenation. Argα141 and Hisβ146 are the C-termini of theα- 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 andhydrogen 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 andthe R group of Argα141. (c) A focus on the salt bridges and hydrogen bonds in which theresidues located at the C-termini of β-chains are involved. All of these links are abolished inthe 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



Bohr Effect II

Carbon dioxide diminishes oxygen binding

• Hydration of CO2 in tissues and extremitiesleads to proton production

• These protons are taken up by Hb as oxygendissociates

• The reverse occurs in the lungs

2,3-Bisphosphoglycerate

An Allosteric Effector of Hemoglobin

• In the absence of 2,3-BPG, oxygenbinding to Hb follows a rectangularhyperbola!

• The sigmoid binding curve is onlyobserved in the presence of 2,3-BPG

• Since 2,3-BPG binds at a site distant fromthe Fe where oxygen binds, it is called anallosteric effector



The structure, in ionic form, of BPG or2,3 -bisphosphoglycerate, an importantallosteric effector for hemoglobin.

2,3-BPG and HbThe "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 frommother



The ionic bindingof BPG to the twoβ-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 ofpH and [BPG].



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 Hbmolecules. 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 bya 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 EFpockets are not involved, although they are present.)

Biomedical Implications

• Myoglobinuria – After massive crushinjury, Mb released from muscle fiberscolors the urine dark red.

• Mb can be detected in plasma ff amyocardial infarction, but assay of serum

enzymes provides a more sensitive indexof myocardial injury.




• Anemias – reduction in the # of RBCs orHb in the blood, can reflect impairedsynthesis of Hb (in iron deficiency) orimpaired production of erythrocytes (infolic acid or Vit B12 deficiency). Diagnosisbegins with spectroscopic measurement ofblood Hb levels.


• Thalassemias – genetic defect resultingfrom the partial or total absence of one ormore alpha or beta chains of Hb.

• Glycosylated Hb (HbA1c) – reliable meansof monitoring blood glucose level



What is an antibody?

• An immunoglobulinthat is capable ofcombining with

specificity to theantigen that elicited itsproduction.

Schematic drawing of animmunoglobulin moleculeshowing the intramolecular andintermolecular disulfide bridges.(A space-filling model of thesame molecule is shown in

Figure 1.11.)



What is an antigen?

• An antigen is any substance that elicits animmune response and is then capable ofbinding to the subsequently producedantibodies.

• Antigens are generally proteins orpolysaccharides, but other substances suchas nucleic acids can also be antigens.

What is an Epitope

• An epitope is the small site on the antigenwhich is recognized by the antibody.

• Usually between one and six sugars oramino acids on the surface of the antigen.

Antibody Uniqueness:

• B-cells produce somewhere between 1 x108

and 1 x 1010 IgG antibodies with differentbinding sites.



Schematic drawing of animmunoglobulin moleculeshowing the intramolecular andintermolecular disulfide bridges.(A space-filling model of thesame molecule is shown in

Figure 1.11.)



Enzyme-Linked Immunosorbent Assay (ELISA)

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