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% a helix (no b structure) Eight helical segments A-H Four terminate in proline Interior: hydrophobic except for two histidines

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

“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

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

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 Ds

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

Hemoglobin: cooperative binding Much better O2 release