Chapter 5: Protein Function

Main functions of globular proteins?

Defense (antibodies, cytokines), storage (myoglobin, ferritin), transport (hemoglobin, serotonin transporter), contraction (actin, myosin), catalysis (chymotrypsin, lysozyme).

What type of interaction governs protein–ligand binding?

Reversible, transient, and based on noncovalent interactions.

Define ligand.

A molecule that binds reversibly to a protein.

Define binding site.

Region on protein where ligand binds via noncovalent forces.

Why is ligand binding reversible?

Because noncovalent interactions can form and break easily.

What defines a binding equilibrium?

Rate of association = rate of dissociation.

Rate constants for binding equilibrium:

Forward (association): ka; reverse (dissociation): kd.

Equilibrium constant expression:

Ka = [PL]/([P][L]); Kd = 1/Ka.

When does equilibrium occur?

When rate of association equals rate of dissociation.

Mathematical relationship for equilibrium:

ka[P][L] = kd[PL].

Define fraction of bound sites (θ).

θ = [L]/(Kd + [L]).

Graphical interpretation of binding:

Hyperbolic curve of θ vs [L].

At half-saturation (θ = 0.5):

[L] = Kd.

What does a smaller Kd indicate?

Higher binding affinity.

What does a large Kd indicate?

Weak binding.

When ligand is a gas, what replaces [L]?

Partial pressure (pO₂)

Thermodynamic relationship between Kd and ΔG°:

ΔG° = RT ln(Kd)

High affinity means what ΔG° sign?

Negative — spontaneous binding.

Lock-and-key model principle:

Ligand and protein are already complementary in shape.

Limitation of lock-and-key model:

Doesn’t account for conformational changes upon binding.

Induced fit model:

Protein and ligand both change shape for better fit.

Why induced fit is correct model:

Allows high affinity and adaptability for multiple ligands.

What determines specificity in binding?

Size, shape, charge, hydrophobic/hydrophilic complementarity.

Why can’t protein side chains bind O₂ directly?

They lack the proper orbital structure.

What metals can bind O₂ but are toxic when free?

Transition metals like Fe and Cu (generate free radicals).

What compound safely binds O₂?

Heme — an organometallic compound.

Why is heme buried in protein?

Prevents oxidation of Fe²⁺ to Fe³⁺.

What is the metal center in heme?

Iron (Fe²⁺).

How many coordination bonds does Fe²⁺ make?

Six — 4 with porphyrin nitrogens, 1 with His F8 (proximal), 1 with O₂.

Which His binds directly to Fe?

His F8 (proximal histidine).

Which His stabilizes O₂ binding?

His E7 (distal histidine).

What residues prevent oxidation of Fe²⁺?

Val and Phe (hydrophobic residues).

What is myoglobin’s primary function?

O₂ storage in muscle.

What is hemoglobin’s primary function?

O₂ transport in blood.

Why is CO toxic?

It binds to Fe²⁺ with much higher affinity than O₂ (~250× stronger in proteins).

How does CO bind Fe²⁺?

Linearly — donates lone pair to Fe d-orbitals.

Why does the protein pocket reduce CO affinity?

Steric hindrance from distal His forces bent binding geometry.

What happens when CO binds too strongly?

O₂ is displaced — inhibiting respiration.

Why does O₂ bind bent?

Allows hydrogen bonding with distal His.

Myoglobin Kd characteristics:

Very low Kd → high affinity → poor O₂ release

Myoglobin binding curve type:

Hyperbolic (noncooperative).

Why is myoglobin unsuitable for transport?

Holds O₂ tightly, releases only at very low O₂ concentrations.

Hemoglobin binding curve type:

Sigmoidal (cooperative).

What does cooperativity mean?

Binding at one site affects affinity at others.

Hemoglobin P₅₀ value:

≈ 26 Torr (partial pressure at 50% saturation).

Myoglobin P₅₀ value:

≈ 2.8 Torr.

Effect of cooperativity on O₂ release:

Enables O₂ pickup at lungs and release at tissues efficiently.

What structure allows cooperativity?

Quaternary structure — multiple subunits

Hemoglobin composition:

4 subunits (2α, 2β).

Positive cooperativity:

First binding increases affinity at remaining sites.

Negative cooperativity:

First binding decreases affinity at remaining sites.

Noncooperative binding example:

Myoglobin

Equation for Hill plot:

log(θ/(1−θ)) = n log(pO₂) − log(Kd).

What is n in Hill equation?

Hill coefficient — measures cooperativity.

n = 1 indicates?

Noncooperative binding.

n > 1 indicates?

Positive cooperativity.

n < 1 indicates?

Negative cooperativity

Max Hill coefficient for Hb?

≈ 3 (cannot fill all 4 sites simultaneously).

What percent of CO₂ binds directly to Hb?

≈ 15–20%.

Where does CO₂ bind on Hb?

Amino termini of globin chains → forms carbamate.

What does carbamate formation do?

Releases H⁺ → lowers pH (Bohr effect).

What is the Bohr effect?

Lower pH and higher CO₂ reduce Hb O₂ affinity → promotes O₂ release.

How is the rest of CO₂ transported?

Converted to bicarbonate (HCO₃⁻) by carbonic anhydrase.

Equation for CO₂ conversion:

CO₂ + H₂O ↔ H⁺ + HCO₃⁻.

Result of H⁺ release:

Favors T-state (low affinity) Hb — encourages O₂ unloading.

What causes cooperativity in Hb?

Conformational changes between T and R states.

What happens to subunits during transition?

α₁β₁ rotates ~15° relative to α₂β₂.

What structural feature is lost in R-state?

Central cavity (“donut hole”)

What is 2,3-BPG?

A negatively charged metabolite regulating Hb function.

Where does 2,3-BPG bind?

Central cavity of T-state Hb.

What charge interactions stabilize BPG binding?

3 positive residues (2 His, 1 Lys) on β-subunits

Effect of BPG binding:

Stabilizes T-state, lowers O₂ affinity, promotes release.

Effect of removing BPG:

Hb favors R-state → higher affinity, less O₂ release.

Why does BPG concentration increase at high altitude?

To enhance O₂ unloading where partial pressure is low.

BPG effect on O₂ curve:

Shifts right → decreased affinity → better tissue oxygenation.

Composition of HbF:

2α + 2γ subunits.

Structural difference in γ subunit:

His → Ser mutation → eliminates BPG binding site.

Effect on O₂ affinity:

Higher (left-shifted curve).

Why does HbF need higher affinity?

To extract O₂ from maternal blood.

Can HbF still switch states?

Yes, but less frequently between T and R states.

Why is heme visible in UV/visible range?

Strong chromophore.

Absorbance of deoxy Hb (Fe²⁺):

429 nm (purple color).

Absorbance of oxy Hb

414 nm (red color).

Color of venous vs arterial blood

Venous — purple (deoxy); arterial — red (oxy).

Spectroscopy use:

Measure O₂ saturation in real time.

Cause of sickle cell mutation:

Glu → Val substitution in β chain.

Effect of sickle cell  mutation:

Nonpolar Val exposed on surface → aggregates with other deoxy Hbs.

Result of aggregation of sickled cells:

Polymerized Hb → sickled red cells.

Why are sickled cells inefficient?

Block capillaries, reduce O₂ transport.

Homozygous sickle cell outcome:

Severe anemia, often fatal in childhood.

Heterozygous sickle cell advantage:

Resistance to malaria.

Two types of immune systems:

Cellular and humoral (“fluid”) immune systems.

Function of cellular immune system:

Destroys infected cells and pathogens inside cells.

Main cellular immune components:

Macrophages, Tc (killer T cells), TH (inflammatory T cells).

Function of humoral immune system:

Targets extracellular pathogens with antibodies.

Main humoral immune components:

B-lymphocytes, helper T cells (TH2).

What is an antigen?

A molecule recognized as foreign that triggers antibody production.

Examples of antigens:

Viral coat proteins, bacterial surface carbs, foreign cell markers.

Define antibody (immunoglobulin)

Protein produced by B-cells that binds antigens with high specificity.