1. Suggested HW Ch. 5 1 – 9 (Chapter 5.1, 5.2)
Note: Protein Explorer (originally due Friday) delayed

2. Kd What is the definition of Kd?
Protein X interacts with Mg2+ with a Kd of 0.5mM. You have a solution of 0.1 mM Protein X. How much Mg2+ should you add so that the equilibrium concentration of complex (X•Mg2+) is 0.08 mM?
You do an experiment to measure the interaction between proteins Y and Z. When a solution of 0.15 pM Y and 0.56 nM Z reaches equilibrium, you determine that the concentration of Y•Z is pM. What is the Kd describing the interaction?

4. Case study: oxygen binding in myoglobin and hemoglobin
Oxygen is poorly soluble in water (blood)
Iron (Fe2+)/O2 complex is soluble
But free iron is toxic
Use proteins containing an iron cofactor
Myoglobin
Hemoglobin

5. Iron is part of a heme prosthetic group: permanent association with protein
“Porphyrin” ring

6. Iron has six coordination sites
Four bind heme nitrogens
One binds protein histidine
“proximal” histidine
His93/HisF8
One can bind O2

7. Structure of myoglobin
Extremely compact
“Globin” family
~75% a helix (no b structure)
Eight helical segments
A-H
Four terminate in proline
Interior: hydrophobic except for two histidines

8. Proximal His coordinates Iron Distal His binds oxygen
His E7
Proximal His coordinates
Iron
Distal His binds oxygen
-increases affinity
-decreases affinity
for carbon monoxide
CO still preferred over O2
-rotation (breathing)
allows O2 exit &
entry
Proximal His
His93
His F8

9. “Globin” fold Six helices: “Three-over-three a-helical sandwich”
Oxygen-carrying molecules
Hemoglobins, myoglobins, cytoglobins, etc
Heme-utilizing enzymes
dehaloperoxidase
Mammals
Worms
Fish
Plants
Bacteria

10. O2 binding by myoglobin Reversible
Myoglobin•O2 ↔ Myoglobin + O2
O2 is a gas: use partial pressures (pO2) instead of molarity
Gas concentration proportional to pressure

11. Myoglobin:
Hyperbolic dependence of O2 binding on pO2

12. Protein flexibility in myoglobin
Structural ‘breathing’ to allow O2 entry
Deoxymyoglobin vs. oxymyoglobin
Change in porphyrin ring, position of iron

14. Why hemoglobin (ie. why not just myoglobin)?
This is where the binding calculations get interesting
Oxygen carrier needs to ‘pick up’ O2 in oxygen-rich (pO2 > 10 kPa) blood surrounding lungs, & ‘drop off’ O2 in oxygen-poor tissues (pO2 ~ 4)
Hyperbolic binding of myoglobin: too insensitive to these types of Ds

15. Myoglobin: good at “picking up” O2, but won’t let go
Tissues
Lungs
Little O2
“Dropped Off”

16. Hemoglobin: cooperative binding
Much better
O2 release