Myoglobin & Hemoglobin

Structure, Function & malfunction of Biomolecules

Primary Structure

Sequence of amino acids in a protein connected via peptide linkage.

Example –the enzyme lysozyme:

1 2 3 4 5 126 127 128 129 Lys-Val-Phe-Gly-Arg...Gly-Cys-Arg-Leu

Note: By convention, amino acid sequences are written starting with the amino terminus.

Secondary Structure

Regular patterns of relatively small segments of a protein held together mainly by H-bonds

α-helix -structure

http://www.ultranet.com/~jkimball/BiologyPages/S/SecondaryStructure.html

Examples:

Tertiary Structure

Overall 3-D shape of a protein. Two basic types are globular and fibrous.

Globular (Pepsin) Fibrous (Collagen)

Examples:

Quaternary Structure

Overall 3-D shape of a multi-subunit protein

Rabbit muscle glycogen phosphorylase

http://bmbiris.bmb.uga.edu/wampler/tutorial/prot4.html

Example:

Protein Function in Cell

1. Enzymes Catalyze biological reactions

2. Structural role Cell wall Cell membrane Cytoplasm

Protein: The Machinery of Life

NH2-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Ser-Ala-Val-Thr-Ala-Leu-Trp-Gly-Lys-Val-Asn-Val-Asp-Glu-Val-Gly-Gly-Glu-…..

Oxygen Transport Proteins Myoglobin

Exhibits Michaelis-Menten properties

Hemoglobin Exhibits allosteric properties

Myoglobin Single polypeptide with

154(human) amino acids

C774H1224N210O222S5

17,183.8 daltons(human)

8 helices (A-H)

Located in skeletal & cardiac muscle

[high] in diving mammals like whale & seals

O2 Binding Curve

Myoglobin has high affinity for O2.

P50 = 2.8 Torr

Allows myoglobin to act as O2 storage reserve.

Releases O2 when pO2 becomes low indicating O2 deprivation.

tissu

es

arte

rial p

ress

ure

pO2 (partial pressure of O2) (Torr)

20 100

satu

ratio

n w

ith O

2

100

50

2.8

N

N N

N

HO O

Fe

Heme (Fe2+) has affinity for O2.

Hematin (Fe3+) cannot bind O2.

Located in crevice where it is protected from oxidation.

Heme Prosthetic Group

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

• CO binds tightly; linear.

• O2 binds less tightly, bent structure.

• Distal His forces bent binding of both, weakens CO binding.

Fe

OO

Fe

CO

Myoblobin:CO complexes

Red Blood Cell (Erythrocyte)

Model Molecule: Hemoglobin

Hemoglobin – Quaternary Structure

Two α (141 AA/ α)subunits and two β (146 AA/ β)subunits

22

Heme

Hemoglobin Structure

Each polypeptide chain resembles myoglobin tertiary structure but 1˚ sequence varies.

Invariant residues indicate importance of those residues in function.

Oxygen Binding Hb exhibits + cooperativity.

Eaton et al. Nature Struct. Biol. 1999, 6, 351

Water bound to heme Iron

O2 Binding to Hemoglobin

Oxygen bound to heme Iron

O2 Binding to Hemoglobin

HbT-state deoxy

Hb R-state - oxy

Hb Variants

http://oregonstate.edu/instruction/bb450/stryer/ch10/Slide27.jpg

HbA2

22

Present in ~2% of adults

Embryonic Hb 22

Has affinity for O2

Fetal Hb 22

Has affinity for O2

Bohr Effect

CO2 pH Some side groups remain

protonated at lower pH.

Stabilizes T state and promotes unloading of O2 to active tissues.

Binding of CO2 also stabilizes T state.

CO2 binds to amino groups. http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG04_50.JPG

2, 3-Bisphosphoglycerate Stabilizes deoxyHb (T

state)

Facilitates unloading of O2 in tissue.

O O

O

O-

P

O

O-

O-

P

O

O-

-O

2, 3-bisphosphoglycerate

pO2 (partial pressure of O2) (Torr)

20 100

satu

ratio

n w

ith O

2

100

50

- BPG

+ BPG

2,3-BPG Binding to Hb

http://oregonstate.edu/instruction/bb450/stryer/ch10/Slide26.jpg

High Altitude and BPG

At higher altitudes, the [BPG] increases allowing Hb to unload O2 more easily.

http://www.bio.davidson.edu/Courses/anphys/1999/Yusi/dpgoxyhbgraph.jpg

Stored Blood & BPG 2,3-BPG becomes depleted in stored blood,

so R state of Hb is stabilized.

If BPG depleted blood is used for a transfusion, the R state Hb doesn’t release O2.

Add inosine to stored blood to maintain BPG levels.

CO Poisoining

CO is “competitive inhibitor” of O2. Affinity is 200X greater than that of O2.

CO also inhibits unloading O2 of in tissues.

Sickle Cell Anemia

Normal red blood cells are round like doughnuts, and they move through small blood tubes in the body to deliver oxygen.

Sickle red blood cells become hard, sticky and shaped like sickles. When these hard and pointed red cells go through the small blood tube, they clog the flow and break apart. This can anemia.

The origin of the disease is a small change in the protein hemoglobin

The change in cell structure arises from a change inthe structure of hemoglobin.

A single change in an amino acid causes hemoglobinto aggregate.

Scanning electron microscopic image of Red bllod cells

Differences in Red Blood Cells

Sickle Cell Hemoglobin

Significant change

in structure caused

by the single mutation

Hemoglobin S

L-Valine(Val / V)

L-Glutamic acid(Glu/ E)

Sickle Cell Hemoglobin

GUG CAC CUG ACU CCU GAG GAG AAGval his leu thr pro glu glu lys 1 2 3 4 5 6 7 8

GUG CAC CUG ACU CCU GUG GAG AAGval his leu thr pro val glu lys 1 2 3 4 5 6 7 8

Mutation (in DNA)

Normal mRNA

Normal protein

Mutant mRNA

Mutant protein

Glutamate (glu), a negatively charged amino acid, is replaced by valine (val), which has no charge.

at 6β