Suggested HW Ch. 5

• 1 – 9 (Chapter 5.1, 5.2)

• Note: Protein Explorer (originally due Friday) delayed

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 0.021 pM. What is the Kd describing the interaction?

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

Iron is part of a heme prosthetic group: permanent association with protein

“Porphyrin” ring

Iron has six coordination sites

Four bind heme nitrogens

One binds protein histidine “proximal” histidine

His93/HisF8

One can bind O2

Structure of myoglobin

• Extremely compact• “Globin” family• ~75% helix (no

structure)– Eight helical segments

• A-H

– Four terminate in proline

• Interior: hydrophobic except for two histidines

Proximal HisHis93His F8

Distal HisHis64His 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

“Globin” fold

Six helices:“Three-over-three helical sandwich”

Oxygen-carrying molecules Hemoglobins, myoglobins, cytoglobins, etc Heme-utilizing enzymes

dehaloperoxidase

Mammals Worms Fish Plants Bacteria

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

502

2

PpO

pO

Myoglobin:

Hyperbolic dependence of O2 binding on pO2

Protein flexibility in myoglobin

• Structural ‘breathing’ to allow O2 entry

• Deoxymyoglobin vs. oxymyoglobin

– Change in porphyrin ring, position of iron

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 s

Tissues Lungs

Little O2 “Dropped Off”

Myoglobin: good at “picking up” O2, but won’t let go

Hemoglobin: cooperative binding

Much betterO2 release