Transcript PPT File

高等生化學
Advanced Biochemistry
Protein Function
陳威戎
Key principles of protein function
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.
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
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.
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 CO2.
12. Oxygen binding to hemoglobin is regulated by 2,3-BPG.
13. Sickle-cell anemia is a molecular disease of hemoglobin.
1. Oxygen can be bound to a heme prosthetic group
2. Myoglobin has a single binding site for oxygen.
The structure of myoglobin
3. Protein-ligand interactions can be described quantitatively
Equilibrium expression of the reversible binding of a protein (P) to
a ligand (L):
Ka: association constant
3. Protein-ligand interactions can be described quantitatively
Kd: dissociation constant
A hypothetical binding curve for a ligand L
A curve describing the binding of oxygen to myoglobin
Some Protein Dissociation Constants
4. Protein structure affects how ligands bind
The binding of oxygen and carbon monoxide to heme
Steric effects on the binding of ligands to the heme of
myoglobin
5. Oxygen is transported in blood by hemoglobin
1. Erythrocytes (red blood cells)
2. Hemocytoblasts
3. Saturation of oxygen in arterial and venous blood
4. Myoglobin (Mb): insensitive to small changes in O2 conc.
oxygen storage
5. Hemoglobin (Hb): highly sensitive, oxygen transport
6. Hemoglobin subunits are structurally similar to Myoglobin
Amino acid sequence comparison for Mb, Hba and Hbb
Dominant interactions between hemoglobin subunits
7. Hemoglobin undergoes a structural change on binding O2
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 a1b2 (and a2b1) interface.
4. Binding of O2 to a Hb subunit in the T state triggers a change in
conformation to the R state, narrowing the pocket between the
b subunits.
Some ion pairs that stabilize the T state of deoxyhemoglobin
Some ion pairs that stabilize the T state of deoxyhemoglobin
The T → R transition
Changes in conformation near heme on O2 binding to
deoxyhemoglobin
8. Hemoglobin binds O2 cooperatively
1. Hb must bind O2 efficiently in the lungs (pO2= 13.3 kPa), and
release O2 in the tissues (pO2= 4 kPa).
2. Hb solves the problem by undergoing a transition from a lowaffinity state ( the T state) to a high-affinity state ( the R state)
as more O2 molecules are bound,
3. Hb has a hybrid S-shaped, or sigmoid, binding curve for O2.
4. 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
A sigmoid (cooperative) binding curve
Structural changes in a multisubunit protein
undergoing cooperative binding to ligand
Structural changes in a multisubunit protein
undergoing cooperative binding to ligand
9. Cooperative ligand binding can be described quantitatively
Equilibrium expression of a protein (P) with n binding sites to a
ligand (L):
Ka: association constant
Hill equation
nH: Hill coefficient ; a measure of the degree of cooperativity
Hill plots for the binding of oxygen to myoglobin
and hemoglobin
Carbon monoxide: a stealthy killer
Carbon monoxide: a stealthy killer
10. Two models suggest mechanisms for cooperative binding
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
1966
Two general models for the interconversion of inactive and
active forms of cooperative ligand-binding proteins
11. Hemoglobin also transports H+ and CO2
Hb carries two end products of cellular respiration - H+ and
CO2 – from the tissues to the lungs and the kidneys.
A reaction catalyzed by carbonic anhydrase. (in erythrocytes)
The binding of O2 by Hb is profoundly influenced by pH and CO2
concentration. ~ Bohr effect
Hb transports about 40% of the total H+ and 15-20% of the CO2.
In peripheral tissues, low pH and high [CO2] → O2 released
In the capillaries of the lung, high pH and low [CO2] → O2 bound
11. Hemoglobin also transports H+ and CO2
In Hb, O2 binds to the iron atom of the hemes.
H+ binds to specific amino acid residues.
CO2 binds as a carbamate to the a-amino group at the Nterminal end of each globin chain.
Effect of pH on the binding of oxygen to hemoglobin
12. O2 binding to Hb is regulated by BPG
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 O2.
BPG binds at a site distant from the O2binding site and regulates the O2-
binding affinity of Hb in relation to the
pO2 in the lungs.
Effect of BPG on the binding of oxygen to hemoglobin
Effect of BPG to deoxyhemoglobin
13. Sickle-cell anemia is a molecular disease of Hb
Comparison of normal and sickle-shaped erythrocytes
Normal and sickle-cell hemoglobin
Normal and sickle-cell hemoglobin
A single a.a. substitution:
Glu6 to Val6 in two b 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.
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.
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.
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 TC 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 TH cells.
MHC proteins
Structure of a human
class I MHC protein
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: a, d, e, g, m ; 2 types of light chain: k and l
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
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
5. The antibody-antigen interaction is the basis for a
variety of important analytical procedures.
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)
Antibody techniques- general method
Antibody techniques- ELISA
III. Protein interactions modulated by chemical energy:
Actin, myosin, and molecular engineering
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.
Motor Proteins underlies~
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.
1. The major protein of muscle are myosin and actin-Myosin
Myosin (Mr 540,000): 6 subunits
2 Heavy chains (each of Mr 220,000)
4 Light chains (each of Mr 20,000)
C-terminus: extended a helices wrapped around each other in
a fibrous, left-handed coiled coil similar to a-keratin.
N-terminus: ATP-binding sites; light chains associated with the
globular domains
Myosin has two heavy chains and two light chains
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
1. The major protein of muscle are myosin and actin-actin
G-actin (globular actin; Mr 42,000): monomeric actin
F-actin (filamentous actin): long polymer of G-actin
Thin filament: F-actin + troponin + tropomyosin
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
2. Additional proteins organize the thin and thick filaments
into ordered structures.
Minor muscle proteins in thin filaments:
a-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
3. Myosin thick filaments slide along
actin thin filaments.
Molecular mechanism of
muscle contraction
Molecular mechanism of muscle contraction
ATP
binding
Molecular mechanism of muscle contraction
ATP
hydrolysis
Molecular mechanism of muscle contraction
Pi release
Molecular mechanism of muscle contraction
ADP
release
Power Stroke
Regulation of muscle contraction
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 Ca2+-binding protein, causes a
conformational change in the complex, exposing the
myosin-binding sites on the thin filaments.
Regulation of muscle contraction
Regulation of muscle contraction