MCB 2000 CH 5: Protein Binding

What are 4 main categories of proteins?

What do they bind?

Enzymes = bind substrates

Receptors = bind hormones

Antibodies = binds invaders

Myoglobin + Hemoglobin = binds oxygen

What is the dissociation constant? What does it indicate?

Dissociation Constant (Kd) → determines tightness of binding

What is molecular recognition?

What is an example of this?

Molecular recognition = ability to pick partner molecule out of lookalike

Estrogen receptor binds to estradiol but NOT testosterone

• Despite similar chemistry

What does a steep hyperbolic curve indicate about relationship b/w receptors and ligand concentration?

What does a less steep hyperbolic curve indicate about relationship b/w receptors and ligand concentration?

Steep hyperbolic curve → receptors = bound very quickly @ low [ligand]

Less steep hyperbolic curve → receptors = bound gradually @ low [ligand]

What is L_1/2?

What does a lower L_1/2 indicate?

Curve touches 100% receptors bound: (True/False)

What is the difference in status of receptors between L_1/2 of individual molecules compared to total molecules?

L_1/2 = [ligand] @ which ½ receptors = bound, ½ receptors = free

Lower L_1/2 → lower [L] needed to reach ½ occupancy → tighter binding

• Lower L_1/2 → higher affinity

False → approaches, BUT never touches

• always some free receptor

L_1/2 of Individual molecules → constantly changing

L_1/2 of Total molecules → stay the same

What is Kd equal to? In concentrations of receptor, ligand, and receptor-ligand complex?

What does Kd equal @ [L] = L_1/2?

Kd = [R][L]/[RL]

@ [L] = L_1/2 → [R] = [RL] → Kd = L_1/2

How many orders of magnitude do the Kd values in biology span?

Describe the 3 types of binding interactions and the associated Kd values

12 orders of magnitude

• Metabolite-protein interactions = 10^-3 mM

• Antibody-antigen = 10^-9 nM to 10^-12 pM

• Avidin-biotin = 10^-15 fM → holds biotin VERY TIGHTLY

  • Avidin = protein that binds to biotin

    • Laboratorial superglue → stick molecules together

What are the 3 main differences between myoglobin (Mb) and hemoglobin (Hb)?

Myoglobin (Mb)

• 1 polypeptide

• Inside muscle cells

• Storage

Hemoglobin (Hb)

• Tetramer = 2 α + 2 β subunits

• RBCs

• Transport (Lungs → Tissues)

What is a heme group?

How does it relate to protein?

Why are heme groups described as prosthetic groups?

How many coordination sites does Fe have? How many of them are occupied by Nitrogens?

• What are the roles of the free sites?

Heme group = ring-shaped iron-containing molecule that binds O₂

Protein = folded around heme group

Prosthetic group = non-protein molecule permanently attached to protein → essential for protein’s function

Fe = 6 coordination sites → 4 occupied by N → 2 free sites

• Top site = O₂ binding site

• Bottom site = Histidine from protein chain

What is the proximal histidine?

What is the role of proximal histidine in relation to heme?

Proximal Histidine = Histidine of protein → Heme Bottom Site

• Physically links protein chain to heme as mechanical unit → important for cooperativity

What is the distal histidine?

What are the 2 primary roles of distal histidine?

Distal Histidine = donates H-bond to LPs of O₂ when bound to Fe

• Stabilizes O₂ complex

• Discriminates against CO

  • CO = binds more strongly to iron than O₂

  • Helps Heme favor O₂ through stabilizing H bonds of LPs

Why does CO bond more strongly to Fe than O₂?

How would this change if distal histidine was absent?

What is the effect of CO binding to one Hb subunit?

CO = binds Fe 200x more tightly than O₂

CO = binds Fe 20,000x more tightly than O₂

Hb shifts toward high-affinity state → O₂ held on tightly

What is neuroglobin?

Neuroglobin = engineered to bind CO 500x more tightly than O₂

• Has lower Kd

• Strips CO off rat Hb administered w/ lethal dose CO

• Neuroglobin-CO complex → excreted

What is the treatment for when CO binds to 1 subunit of Hb?

Flood patient w/ 100% O₂ → displace CO

What are the differences in types of curves between myoglobin (Mb) and hemoglobin (Hb)?

What do they indicate about each type of protein?

What is the x and y-axis of the graph?

Myoglobin → hyperbolic

• Saturates quickly

• Holds tight → releases @ low pO₂

• Storage protein

Hemoglobin → sigmoidal

• Saturates slowly

• High saturation (lungs) → low saturation (tissues)

• Transport protein

X-axis = pO₂

Y-axis = fractional saturation

What is P₅₀?

What does it indicate?

P₅₀ = pO₂ @ which protein is ½ saturated → protein affinity

• Lower P₅₀ → Tighter binding

• Higher P₅₀ → Looser binding

What is the relationship between P₅₀ and Kd in myoglobin?

P₅₀ = Kd

What is the difference between T and R state?

What are the differences in Fe and Salt Bridge status in both states?

T (Tense) state = none of 4 binding

• Fe = below plane of porphyrin ring → bad binding geometry

• Salt Bridge = hold tetramer in place = protein locked

R (Relaxed) state = enough O₂ is bound

• Fe = up in plane of porphyrin ring → good binding geometry

• Salt Bridge = broken b/w subunits = protein unlocked