Advanced Biochemistry 高等生化學 Protein Function 陳威戎

1. The functions of many proteins involve the reversible binding of other molecules. 2. A ligand binds at a site on the protein called the binding site, which is complementary to the ligand. 3. Proteins are flexible. 4. The binding of a protein and ligand is often coupled to a conformational change. 5. Interactions between ligands and proteins may be regulated. Key principles of protein function

1.Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins 2.Complementary Interactions between Proteins and Ligands: The Immune System and Immunoglobulins 3.Protein Interactions Modulated by Chemical Energy: Actin, Myosin, and Molecular Motors Protein Function

1. Oxygen can be bound to a heme prosthetic group. 2. Myoglobin has a single binding site for oxygen. 3. Protein-ligand interactions can be described quantitatively. 4. Protein structure affects how ligands bind. 5. Oxygen is transported in blood by hemoglobin. 6. Hemoglobin subunits are structurally similar to myoglobin. 7. Hemoglobin undergoes a structural change on binding oxygen. 8. Hemoglobin binds oxygen cooperatively. 9. Cooperative ligand binding can be described quantitatively. 10. Two models suggest mechanisms for cooperative binding. 11. Hemoglobin also transports H + and CO Oxygen binding to hemoglobin is regulated by 2,3-BPG. 13. Sickle-cell anemia is a molecular disease of hemoglobin. I. Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins

1. Oxygen can be bound to a heme prosthetic group

2. Myoglobin has a single binding site for oxygen.

The structure of myoglobin

Equilibrium expression of the reversible binding of a protein (P) to a ligand (L): K a : association constant 3. Protein-ligand interactions can be described quantitatively

K d : dissociation constant 3. Protein-ligand interactions can be described quantitatively

A hypothetical binding curve for a ligand L

A curve describing the binding of oxygen to myoglobin

Some Protein Dissociation Constants

The binding of oxygen and carbon monoxide to heme 4. Protein structure affects how ligands bind

Steric effects on the binding of ligands to the heme of myoglobin

1. Erythrocytes (red blood cells) 2. Hemocytoblasts 3. Saturation of oxygen in arterial and venous blood 4. Myoglobin (Mb): insensitive to small changes in O 2 conc. oxygen storage 5. Hemoglobin (Hb): highly sensitive, oxygen transport 5. Oxygen is transported in blood by hemoglobin

6. Hemoglobin subunits are structurally similar to Myoglobin

Amino acid sequence comparison for Mb, Hb  and Hb 

Dominant interactions between hemoglobin subunits

1. Two major conformations of hemoglobin: R (relaxed) state and T (tense) state 2. T state is more stable (deoxyhemoglobin) 3. T state is stabilized by a greater number of ion pairs, many of which lie at the  1  2 (and  2  1 ) interface. 4. Binding of O 2 to a Hb subunit in the T state triggers a change in conformation to the R state, narrowing the pocket between the  subunits. 7. Hemoglobin undergoes a structural change on binding O 2

Some ion pairs that stabilize the T state of deoxyhemoglobin

The T → R transition

Changes in conformation near heme on O 2 binding to deoxyhemoglobin

1. Hb must bind O 2 efficiently in the lungs (pO 2 = 13.3 kPa), and release O 2 in the tissues (pO 2 = 4 kPa). 2. Hb solves the problem by undergoing a transition from a low- affinity state ( the T state) to a high-affinity state ( the R state) as more O 2 molecules are bound, 3. Hb has a hybrid S-shaped, or sigmoid, binding curve for O Allosteric protein: binding of a ligand to one site affects the binding properties of another site on the same protein. 5. Modulators: inhibitors or activators; homotropic or heterotropic 8. Hemoglobin binds O 2 cooperatively

A sigmoid (cooperative) binding curve

Structural changes in a multisubunit protein undergoing cooperative binding to ligand

Equilibrium expression of a protein (P) with n binding sites to a ligand (L): K a : association constant Hill equation n H : Hill coefficient ; a measure of the degree of cooperativity 9. Cooperative ligand binding can be described quantitatively

Hill plots for the binding of oxygen to myoglobin and hemoglobin

Carbon monoxide: a stealthy killer

Concerted model (MWC model): proposed by Jacques Monod, Jefferies Wyman, and Jean-Pierre Changeux in 1965 Sequential model: proposed by Daniel Koshland and colleagues in Two models suggest mechanisms for cooperative binding

Two general models for the interconversion of inactive and active forms of cooperative ligand-binding proteins

Hb carries two end products of cellular respiration - H + and CO 2 – from the tissues to the lungs and the kidneys. A reaction catalyzed by carbonic anhydrase. (in erythrocytes) The binding of O 2 by Hb is profoundly influenced by pH and CO 2 concentration. ~ Bohr effect Hb transports about 40% of the total H + and 15-20% of the CO 2. In peripheral tissues, low pH and high [CO 2 ] → O 2 released In the capillaries of the lung, high pH and low [CO 2 ] → O 2 bound 11. Hemoglobin also transports H + and CO 2

In Hb, O 2 binds to the iron atom of the hemes. H + binds to specific amino acid residues. CO 2 binds as a carbamate to the  -amino group at the N- terminal end of each globin chain. 11. Hemoglobin also transports H + and CO 2

Effect of pH on the binding of oxygen to hemoglobin

The interaction of 2,3-bisphosphoglycerate (BPG) with Hb provides an example of heterotropic allosteric modulation. BPG is known to greatly reduce the affinity of Hb for O 2. BPG binds at a site distant from the O 2 - binding site and regulates the O 2 - binding affinity of Hb in relation to the pO 2 in the lungs. 12. O 2 binding to Hb is regulated by BPG

Effect of BPG on the binding of oxygen to hemoglobin

Effect of BPG to deoxyhemoglobin

Comparison of normal and sickle-shaped erythrocytes 13. Sickle-cell anemia is a molecular disease of Hb

Normal and sickle-cell hemoglobin

A single a.a. substitution: Glu 6 to Val 6 in two  chains HbS has two fewer negative charges than HbA. Creates a “sticky” hydrophobic contact point on the outer surface, causes deoxyHbS to associate abnormally with each other, forming the long, fibrous aggregates.

1. The immune response features a specialized array of cells and proteins. 2. Self is distinguished from nonself by the display of peptides on cell surfaces. 3. Antibodies have two identical antigen-binding sites. 4. Antibodies bind tightly and specifically to antigen. 5. The antibody-antigen interaction is the basis for a variety of important analytical procedures. II. Complementary Interactions between Proteins and Ligands: The Immune System and Immunoglobulins

1. The immune response features a specialized array of cells and proteins.

2. Self is distinguished from nonself by the display of peptides on cell surfaces. MHC (major histocompatibility complex) proteins Class I MHC Each individual produces up to 6 class I MHC variants. Bind and display peptides derived from cellular proteins. Recognition targets of the T-cell receptors of the T C cells. Class II MHC Occur on the surfaces of macrophages and B lymphocytes. Each human produces up to 12 variants. Bind and display peptides derived from external proteins. Recognition targets of the T-cell receptors of the T H cells.

MHC proteins

Structure of a human class I MHC protein

3. Antibodies have two identical antigen-binding sites. Five classes of immunoglobulins: 5 types of heavy chain:  ; 2 types of light chain:  and IgD, IgE: overall structures similar to that of IgG IgE: allergic response; interacts with basophils and mast cells. IgM: a cross-linked pentamer, first Ab made by B lymphocytes, major Ab in the early stages of primary immune response. IgA: found in secretions such as saliva, tears, and milk, can be a monomer, dimer or trimer. IgG: major Ab in secondary immune responses, initiated by memory B cells ; most abundant Ab in the blood.

The structure of immunoglobulin G

Binding of IgG to an antigen

IgM pentamer of immunoglobulin units

Phagocytosis of an antibody-bound virus by a macrophage

4. Antibodies bind tightly and specifically to antigen- induced fit

Two types of antibodies preparations are in use: Polyclonal antibodies Monoclonal antibodies: by Köhler and Milstein, 1975 Practical uses of antibodies: Affinity column ELISA (enzyme-linked immunosorbent assay) Immunoblot assay (Western blot) 5. The antibody-antigen interaction is the basis for a variety of important analytical procedures.

Antibody techniques- general method

Antibody techniques- ELISA

1. The major proteins of muscle are myosin and actin. 2. Additional proteins organize the thin and thick filaments into ordered structures. 3. Myosin thick filaments slide along actin thin filaments. III. Protein interactions modulated by chemical energy: Actin, myosin, and molecular engineering

1. Contraction of muscles Myosin ; Actin ; additional proteins 2. Migration of organelles along microtubules Kinesins ; Dyneins 3. Rotation of bacterial flagella Rotary motor complex (Proton turbine) 4. Movement of some proteins along DNA Helicases, polymerases, etc. Motor Proteins underlies~

Myosin (M r 540,000): 6 subunits 2 Heavy chains (each of M r 220,000) 4 Light chains (each of M r 20,000) C-terminus: extended  helices wrapped around each other in a fibrous, left-handed coiled coil similar to  -keratin. N-terminus: ATP-binding sites; light chains associated with the globular domains 1. The major protein of muscle are myosin and actin-Myosin

Myosin has two heavy chains and two light chains

Cleavage with trypsin and papain separates the myosin heads from the tails

Ribbon representation of myosin S1 fragment

The major components of muscle- myosin Thick filament

G-actin (globular actin; M r 42,000): monomeric actin F-actin (filamentous actin): long polymer of G-actin Thin filament: F-actin + troponin + tropomyosin 1. The major protein of muscle are myosin and actin-actin

The major components of muscle- F-actin Thin filament

Actin filament bound with myosin head

Structure of skeletal muscle- muscle fibers

Relaxed and contracted muscle

Minor muscle proteins in thin filaments:  -actinin ; desmin ; vimentin ; nebulin Minor muscle proteins in thick filaments: paramyosin ; C-protein ; M-protein ; titins Molecular rulers that regulates the length of the thin and thick filaments: nebulin and titin. Titin extends from the Z disk to the M line. 2. Additional proteins organize the thin and thick filaments into ordered structures.

Muscle contraction

Sarcomere- Contracting

Molecular mechanism of muscle contraction 3. Myosin thick filaments slide along actin thin filaments.

Molecular mechanism of muscle contraction ATP binding

Molecular mechanism of muscle contraction ATP hydrolysis

Molecular mechanism of muscle contraction P i release

Molecular mechanism of muscle contraction ADP release

Power Stroke

Interactions between actin and myosin must be regulated so that contraction occurs only in response to appropriate signals form the nervous system. Regulation is mediated by a complex of two proitens: tropomyosin: binds to the thin filament, blocking the attachment sites for the myosin head groups. troponin: a Ca 2+ -binding protein, causes a conformational change in the complex, exposing the myosin-binding sites on the thin filaments. Regulation of muscle contraction