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 Fe 2+ (ferrous state) 6 coordination bonds for Fe 2+  4 N in porphyrin ring  Electron donating character: prevent oxidation of Fe 2+ to Fe 3+ (ferric state)  2 perpendicular to the prophyrin  1 occupied with proximal His residue  1 binding site for oxygen  Fe 2+ ; 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 myoglobin  Oxygen transport in muscle  Abundant in diving mammals; seals and whales Structure  153 a.a. protein belongs to globin family  8  helical segments  1 heme molecule

Protein-Ligand Interactions : K a

Protein-Ligand Interactions : K d Binding of O 2 to myoglobin   =  [O 2 ] / ([O 2 ] + K d )  = [O 2 ] / ([O 2 ] + [O 2 ] 0.5 ) = [O 2 ] / ([O 2 ] + P 50 ) P 50 : local partial pressure of O 2 at [O 2 ] 0.5

Protein Structure Affects How Ligands Bind O 2 and CO binding to heme  Binding to free heme  CO has more than 20,000 times higher affinity than O 2  Binding to heme in myoglobin  CO has 200 times higher affinity than O 2  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 O 2 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 O 2  In venous blood: 64% are saturated with O 2  Very sensitive to O 2 concentration  Good for O 2 transport Myoglobin  Relatively insensitive to O 2 concentration  Good for O 2 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 O 2 binding  Deoxyhemoglobin  More ion pairs at  1  2 (  2  1) interface  Slightly puckered porphyrin R (relaxed) state : high affinity O 2 binding  O 2 binding state  Planar porphyrin

T and R State of Hemoglobin

Cooperative Binding of Oxygen to Hemoglobin Roles of hemoglobin  In the lung (pO 2 = 13.3 kPa) : binding to O 2  In the tissues (pO 2 = 4 kPa) : releasing O 2 Cooperative binding of O 2 to hemoglobin  Transition form T state to R state upon O 2 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 (n H, Hill coefficient)  Degree of cooperativity  n H = 1 : no cooperativity  n H >1 : positive cooperativity  n H = n : theoretical upper limit, Simultaneous binding of the entire binding sites

Hill Plot for O 2 Binding to Myoglobin and Hemoglobin Log (  /1-  ) = nlog [L] – log K d Log (  /1-  ) = nlog pO 2 – nlog P 50

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 CO 2 by Hemoglobin Transport H + and CO 2 from the tissue to the lungs and kidneys Carbonic anhydrase in erythrocyte  Hydration of CO 2 to form bicarbonate  CO 2 + H 2 O H + + HCO 3 - Bohr effect (1904)  Effect of [CO 2 ] and [H + ] on binding & releasing of O 2 binding by hemoglobin  H + binding : His146 in  subunit and other a.a residues  stabilization of T state  HHb + + O 2 HbO 2 + H +  CO 2 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 O 2 binding affinity of hemoglobin HbBPG + O 2 HbO 2 + BPG (inverse relation) Fetal hemoglobin   2  2   subunits have lower affinity for BPG  High affinity to O 2  Effective extraction of O 2 from its mother’s blood Oxygen Binding to Hemoglobin is Regulated by BPG

 Facilitate O 2 release in the tissue under low pO 2 (high altitudes, hypoxia) Fetal hemoglobin   2  2   subunits have lower affinity for BPG  High affinity to O 2  Effective extraction of O 2 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  K d value; ~ M

Immunoglobulins Ig A Monomer/d imer/trimer Saliva, tear, milk Ig D Unclear function Ig E Allergic response Histamine secretion from mast cells Ig G Major Ab for 2 nd immune response Ig M Monomer/penta mer 1 st Ab from B cells Major Ab for early stage of immune response

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