Chapter 5

Protein Function

Interaction of Proteins with Other Molecules

Ligand A molecule binding reversibly to a protein Other proteins, or any kind of molecules

Binding site for a ligand Complementary to the ligand in size, shape, charge, and

hydrophobic/~philic properties Specific & selective to one or a few ligands

Conformational change of proteins Subtle change (breathing)

Molecular vibrations, small movement of a.a. residues Dramatic change

Movement of major segment of a protein Induced fit

Structural adaptation permitting tighter binding Conformational signal

Cooperativity between ligand and protein interactions

5.1 Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins

Heme

Prosthetic group of oxygen-transporting proteins

Myoglobin, hemoglobin, cytochromes Complex organic ring structure; Protoporphyrin Protoporphyrin with Fe2+ (ferrous state)

6 coordination bonds for Fe2+

4 N in porphyrin ring Electron donating character: prevent oxidation of Fe2+ to Fe3+

(ferric state)

2 perpendicular to the prophyrin 1 occupied with proximal His residue 1 binding site for oxygen

Fe2+ ; oxygen binding Changing from dark purple to bright red color Higher affinity to CO and NO

Heme

Porphyrins

4 Pyrrole rings

Myoglobin (Mb)

Roles of myoglobinOxygen transport in

muscleAbundant in diving

mammals; seals and whales

Structure153 a.a. protein belongs

to globin family8 helical segments 1 heme molecule

Protein-Ligand Interactions : Ka

Protein-Ligand Interactions : Kd

Binding of O2 to myoglobin

=[O2] / ([O2] + Kd)

= [O2] / ([O2] + [O2]0.5)

= [O2] / ([O2] + P50 )

P50 : local partial pressure of O2 at [O2]0.5

Protein Structure Affects How Ligands Bind

O2 and CO binding to heme Binding to free heme

CO has more than 20,000 times higher affinity than O2

Binding to heme in myoglobin CO has 200 times higher affinity than O2

Steric hindrance restricts CO binding

Roles of breathing Heme is deeply buried inside of the

protein Rotation of distal His (10-9 sec) provides

cavities for O2 entrance

Hemoglobin

Red blood cells Generated form hemocytoblast stem cells Hemoglobin production & carrying Loss of intracellular organelles Life time 120 days

Hemoglobin In arterial blood: 96% are saturated with O2

In venous blood: 64% are saturated with O2

Very sensitive to O2 concentration

Good for O2 transport

Myoglobin Relatively insensitive to O2 concentration

Good for O2 storage

Hemoglobin

Structure 2 (141 a.a.), 2 (146 a.a.) chains, and 4 heme groups Globin family of proteins

, chains and myoglobin Low sequence similarity but high structural similarity

Strong interactions between and chains >30 residues are involved Mostly hydrophobic interactions

Structural Change of Hemoglobin upon Oxygen Binding

T (tense) state : low affinity O2 binding Deoxyhemoglobin More ion pairs at 12 (21) interface Slightly puckered porphyrin

R (relaxed) state : high affinity O2 binding O2 binding state Planar porphyrin

T and R State of Hemoglobin

Cooperative Binding of Oxygen to Hemoglobin

Roles of hemoglobin

In the lung (pO2 = 13.3 kPa) : binding to O2

In the tissues (pO2 = 4 kPa) : releasing O2

Cooperative binding of O2 to hemoglobin

Transition form T state to R state upon O2 binding induction of conformational change of the adjacent subunit to R state

Sigmoid binding curve

Allosteric Protein

Allosteric protein Binding of a ligand to one site

affects the binding properties of another site on the same protein

Modulator : activator or inhibitor Homotropic

Modulator = the normal ligand

Heterotropic Modulator ≠ the normal ligand

Cooperative binding (hemoglobin) Allosteric binding in multimeric

proteins Sigmoid binding curve Sensitive to ligand concentration Binding site in stable segment

next to unstable segment

Quantitative Description of Cooperative Ligand Binding

Hill plot; Log (/1-) vs. log [L]

Slope (nH, Hill coefficient) Degree of cooperativity nH = 1 : no cooperativity nH >1 : positive cooperativity nH = n : theoretical upper limit,

Simultaneous binding of the entire binding sites

Hill Plot for O2 Binding to Myoglobin and Hemoglobin

Log (/1-) = nlog [L] – log Kd Log (/1-) = nlog pO2 – nlog P50

Models for Cooperative Binding

MWC model (concerted model) Jaques Monod, Jeffries

Wyman, Jean-Pierre Changeux (1965)

All proteins in the same conformation

Transition to high affinity conformation upon ligand binding

Sequential model Daniel Koshland (1966) Ligand binding induces

conformational change in an individual subunit Induce a similar change in

an adjacent subunit

Transport of H+ and CO2 by Hemoglobin

Transport H+ and CO2 from the tissue to the lungs and kidneys

Carbonic anhydrase in erythrocyte Hydration of CO2 to form bicarbonate CO2 + H2O H+ + HCO3-

Bohr effect (1904) Effect of [CO2] and [H+] on binding &

releasing of O2 binding by hemoglobin H+ binding : His146 in subunit and

other a.a residues stabilization of T state HHb+ + O2 HbO2 + H+

CO2 binding : Forms carbamate group by binding to N terminal amino group Generation of H+

Stabilization of T state by salt bridge

2,3 bisphosphoglcerate (BPG) Abundant in erythrocyte Heterotropic allosteric modulator Binding to cavity between subunits in the T state

Interaction with positive a.a, stabilizing T state,1 BPG/Hb tetramer

Reduced O2 binding affinity of hemoglobin

HbBPG + O2 HbO2 + BPG (inverse relation)

Fetal hemoglobin 22 subunits have lower affinity for BPG High affinity to O2

Effective extraction of O2 from its mother’s blood

Oxygen Binding to Hemoglobin is Regulated by BPG

Oxygen Binding to Hemoglobin is Regulated by BPG

Facilitate O2 release in the tissue under low pO2 (high altitudes, hypoxia)

Fetal hemoglobin 22 subunits have lower affinity for BPG High affinity to O2

Effective extraction of O2 from its mother’s blood

Sickle-Cell Anemia

Hemoglobin S

Glu 6 to Val mutation in two chain (homozygote) Heterozygote has a mild

symptom

Aggregation of deoxygenated hemoglobins by hydrophobic interactions

fiber formation

Sickle-Cell Anemia

Sickle shaped erythrocytes Fragile : lower hemoglobin content Blocking capillaries

5.2 Complementary interaction;The immune system and immunoglobulins

Immune cells

Leukocytes (white blood cells) Recognition & binding to molecules for infection signals

Immune responses

Humoral immune system Bacteria or virus infections Antibodies (immunoglobulins; Ig) mediation

Binding to bacteria, viruses, other foreign molecules destruction Produced from B lymphocytes (B cells)

Cellular immune system Removal of infected cells & parasites/foreign tissues T lymphocytes; cytotoxic T cells (killer T cells)

T-cell receptor-mediated recognition of infected cells or parasites

Helper T cells

Structural properties of antibodies

Immunoglobulin G (IgG) Major class of antibody 4 polypeptide chains; 2 heavy chains + 2 light chains (noncovalent & disulfide bonds) Y-shaped complex; Fa + Fab (antigen-binding fragments)

Structural properties of antibodies

Specificity between antigen and binding sites Shape & location of noncovalent interactions Conformational changes complete interactions Kd value; ~ 10-10 M

Immunoglobulins

Phagocytosis of Ig G-bound virus by macrophage

Antibody techniques

Enzyme-linked immunosorbent assay

5.3 Protein interaction modulated by chemical energy

Contractile force generation in muscle by myosin and actin

Myosin & actin

Skeletal muscle

Molecular mechanism of muscle contraction