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