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 Examples: -structure α-helix http://www.ultranet.com/~jkimball/BiologyPages/S/SecondaryStructure.html

Tertiary Structure Overall 3-D shape of a protein. Two basic types are globular and fibrous. Examples: Fibrous (Collagen) Globular (Pepsin)

Quaternary Structure Overall 3-D shape of a multi-subunit protein Example: Rabbit muscle glycogen phosphorylase http://bmbiris.bmb.uga.edu/wampler/tutorial/prot4.html

Protein Function in Cell Enzymes Catalyze biological reactions 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 a helices (A-H) Located in skeletal & cardiac muscle [high] in diving mammals like whale & seals

pO2 (partial pressure of O2) (Torr) 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. tissues arterial pressure pO2 (partial pressure of O2) (Torr) 20 100 saturation with O2 50 2.8

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

Oxygen Binding to Myoglobin distal histidine 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. proximal histidine http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG04_44.JPG

Myoblobin:CO complexes CO binds tightly; linear. O2 binds less tightly, bent structure. Distal His forces bent binding of both, weakens CO binding. Fe C O Fe O

Red Blood Cell (Erythrocyte)

Model Molecule: Hemoglobin

Hemoglobin – Quaternary Structure a2b2 Two α (141 AA/ α)subunits and two β (146 AA/ β)subunits

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

O2 Binding to Hemoglobin Water bound to heme Iron

O2 Binding to Hemoglobin Oxygen bound to heme Iron

Hb T-state deoxy

Hb R-state - oxy

Hb Variants HbA2 Embryonic Hb Fetal Hb a2d2 Present in ~2% of adults Has  affinity for O2 Fetal Hb a2g2 http://oregonstate.edu/instruction/bb450/stryer/ch10/Slide27.jpg

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 a 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. 100 - BPG saturation with O2 50 + BPG 20 100 pO2 (partial pressure of O2) (Torr)

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 in the structure of hemoglobin. A single change in an amino acid causes hemoglobin to aggregate. a b

Scanning electron microscopic image of Red bllod cells

Differences in Red Blood Cells

Sickle Cell Hemoglobin Hemoglobin S Significant change in structure caused by the single mutation

L-Glutamic acid (Glu/ E) L-Valine (Val / V)

Sickle Cell Hemoglobin at 6β Normal mRNA GUG CAC CUG ACU CCU GAG GAG AAG val his leu thr pro glu glu lys 1 2 3 4 5 6 7 8 Normal protein Mutation (in DNA) Mutant mRNA GUG CAC CUG ACU CCU GUG GAG AAG val his leu thr pro val glu lys 1 2 3 4 5 6 7 8 Mutant protein Glutamate (glu), a negatively charged amino acid, is replaced by valine (val), which has no charge.