Download Chapter 5 Protein Function

Chapter 5 Protein Function
Dr. Rita P.-Y. Chen
陳佩燁博士
中央研究院生化所
Multifunctions of proteins
•Catalyse biochemical reactions (enzyme)
•Transduce signals (G-protein)
•Control life and growth (hormone and receptor)
•Provide protection (hair)
•Provide mobility (muscle)
•Transport materials (hemoglobin)
•Recognize foreign material (immunoglobulin)
•Induce disease (prion)
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Protein-ligand binding
A molecule bound reversibly by a protein is called a ligand.
A ligand binds at a site on the protein called the binding
site.
Ligand may be any kind of molecule.
The protein-ligand interaction is specific.
A protein may have separate binding sites for several
ligands
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Oxygen-binding protein
Oxygen is poorly soluble
in aqueous solution.
How can oxygen arrive
in tissues?
Hemoglobin: transport
oxygen and CO; tetramer,
2 a, 2 b subunits
Myoglobin: oxygen
storage, single
polypeptide of 153
amino acid residues
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Why can they bind oxygen?
Prosthetic group: heme
Prosthetic group: A metal ion or an organic
compound (other than an amino acid) that is
covalently bound to a protein and is
essentially to its activity
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Heme: protoporphyrin,
Fe2+
1. N atom has electron
donating character which
prevents the conversion of
Fe2+ to Fe3+
2. Iron in Fe2+ state binds
oxygen reversibly; in the
Fe3+ state it does not bind
oxygen
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When oxygen binds, the electronic
properties of heme iron change, so blood
change color from dark purple to bright red
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Hemoglobin subunits are structurally
similar to Myoglobin
Hemoglobin has 2 a, 2 b
subunits; less then half are
sequence identical; but 3Dstructures of a and b subunits
are similar.
Only 27 amino acids are
identical between myoglobin
and hemoglobin subunits, but
their structures are very
similar.
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Ka is an association constant
Ka has units of M-1;
The association constant provides a measure of the affinity
of the ligand L for the protein.
a higher value of Ka corresponds to a higher affinity of the
ligand for the protein.
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The ratio of bound to free protein is directly
proportional to the concentration of free ligand
When the concentration of the ligand is much
greater than the concentration of ligand-binding
sites, the binding of the ligand by the protein does
not appreciably change the concentration of free
(unbound) ligand —that is, [L] remains constant.
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Now, use q(theta) to represent the ratio of
ligand binding sites on the protein that are
occupied by ligand
Because [PL] = Ka [L] [P]
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Plot of q versus [L]
Hyperbola 雙曲線
When half of sites are bound (q=0.5), 2[L] = [L] + 1/ Ka,
此時 [L] = 1/Ka = Kd
Kd : dissociation constant 解離常數
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Kd = 1/ Ka
Kd is given in units of molar concentration
(M)
a lower value of Kd corresponds to a higher
affinity of ligand for the protein.
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1 atm = 101.325 kPa
4 kPa in tissue
13.3 kPa in lung
0.26 kPa
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Why is CO toxic?
To free heme Kd(CO) = 1/20000 Kd(O2)
To myoglobin Kd(CO) = 1/200Kd(O2) due
to steric hinderance
How can gas enter the cavity?
Molecular motion of proteins
(breathing)
On a nanosecond time scale
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緊接的
遠端的
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Evolution meaning……..
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Hemoglobin: oxygen transportation
hemocytoblast
Cels with lots of hemoglobin
maturation
Cells lose intracellular organelles :
nucleus, mitochondria, endoplasmic reticulum
Erythrocytes (red blood cells)
Which survive only 120 days
Contain ~34 % hemoglobin
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Hemoglobin 96 % saturated
with O2
Hemoglobin
64 % saturated
with O2
Each 100 ml blood
releases 6.5 ml
oxygen
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Hemoglobin binds oxygen cooperatively
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Hemoglobin has two conformational states
O2
tense
relaxed
Oxygen binding state
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T state is stabilized by
ionic interactions, so T
state is stable without
oxygen binding
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Why does oxygen binding induce T to R conformational
change?
O2
Puckered
縮攏
More planar
when heme binds O2 , porphyrin forms more planar conformation
then shifts helix F
ab subunits pairs slide past each other and
rotate,narrowing the pocket between b subunits
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High O2 affinity
in lung, bind O2
Sigmoid binding curve
of hemoglobin
Low O2 affinity in
tissue, release O2
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Hemoglobin has 4 subunits, O2 binding to
individual subunits can alter the affinity to
O2 in adjacent subunits Modulator (inhibitor or activator)
Binding of a ligand to one site affects the
binding properties of another site on the
same protein
allosteric protein
When the normal ligand and modulator are
identical
homotropic;
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if not,
heterotropic
Hill equation
slope
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Hill plot
Experimental
determined slope
reflects not the
binding sites but the
degree of interaction
between them.
The slope of Hill
plot is denoted by nH
(Hill coefficient),
representing degree
of cooperativity
nH = 1 not cooperative
nH > 1 positive cooperative
nH < 1 negative cooperative
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Two model explain hemoglobin cooperativity: MWC model
(concerted model;all-or-none model), sequential model
一致的
連續的
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Other modulators for hemoglobin : H+, CO2, 2,3bisphosphoglycerate (BPG) reduce the affinity of
hemoglobin for oxygen
Hemoglobin also carry 40 %H+ and 1520% CO2 from tissue to lung and kidney to
excrete them from body
CO2 is not very soluble
Catalyzed by carbonic anhydrase in earthrocyte
Bohr effect:
the effect of pH and CO2 on binding and
release of oxygen by hemoglobin
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H+ binds to several residues , such as
His146 (His HC3)
Low pH, low
oxygen binding
T state is stabilized by
ionic interactions.
Protonated His helps
stabilize T state (low
oxygen affinity)
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CO2 binds to the a-amino group at
the N-terminal end of each subunit,
forms carbaminohemoglobin
It can form additional salt bridges
that help to stabilize T state and
promote oxygen release
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2,3-bisphosphoglycerate (BPG)
BPG has high content in
erythrocytes
BPG binds in the cavity between b
subunits in the T state
T state
R state
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BPG is important
for delivering O2 to
tissues when people
is in high altitudes
If BPG is too high,
it causes Hypoxia
(組織缺氧)
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Fetus needs to get oxygen from mother’s
blood
Fetus has a2g2 hemoglobin (HbF)
a2g2 has lower affinity for BPG
higher affinity for O2
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Sickle-cell Anemia
It’s a genetic disease (each person has two alleles)
Genetic variant in hemoglobin
hemoglobin S
Glu6 Val mutation in b chains (hydrophobic group)
Forms immature red blood cells
sickle cell
Hemoglobin in blood is only half of the normal amount
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When hemoglobin S is
deoxygenated, it
becomes insoluble and
forms polymers
The insoluble fibers are
responsible to the sickle
shape of erythrocyte
Sickle-cell anemia is more
common in Africa
result of
natural selection of malaria
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Protein-ligand interaction: Antibody
and Antigen in immune system
Leukocytes (white blood cells)
macrophage
lymphocytes
T cells
Cytotoxic T cell
(Tc cell)
Helper T cells
(TH cells)
Cellular immune system
B cells
Produce antibody
(immunoglobulin; Ig)
Humoral immune system
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Humoral (體液的)immune system is
directed at bacterial infection and
extracellular virus in body fluid, also
respond to the proteins produces in these
organism.
Cellular immune system destroys hosts
infected by viruses, some parasites, and
foreign tissues 與器官移植的排斥有關
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Mature in
bone marrow
Mature in thymus
HIV 會與 TH cells 結合,
破壞免疫能力
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Any molecule or pathogen capable of eliciting an
immune response is called an antigen
Part of the antigen which can be bound by
antibody or T cell receptor is called antigenic
determinant or epitope.
Molecules of Mr < 5000 are generally not
antigenic. They can be covalently attached to
large proteins to elicit an immune responses. They
were called hapten .『免疫』附著素; 半抗原
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MHC (major histocompatibility complex) proteins
敵我辨識
Detection of protein antigens in the host is
mediated by MHC
MHC proteins bind peptide fragments of
protein digested in the cell and present them
on the outside surface of the cell
Two classes of MHC proteins: class I, class
II
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Occur on the surface of
vertebrate cells; polymorphic
Therefore, it causes tissue
rejection in organ transplanation
Occur on the surface of a
few types of cells such as
macrophage and B cells;
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polymorphic
The maturation of Tc cells in
the thymus includes a stringent
selection process that
eliminates more than 95% of
the developing Tc cells,
including those that might
recognize and bind class I
MHC proteins displaying
peptides from cellular proteins
of the organism itself.
Each survived Tc cell has a
single type of T-cell receptor
that bind to one particular
chemical structure
Human class I MHC protein binds antigen
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Helper T cells (TH)
TH cells works indirectly
TH cells stimulate the selectively
proliferation of those Tc and B cells that
bind to a particular antigen ----clonal
selection
HIV binds TH cells ------AIDS
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B cells produce 5 classes
antibodies:IgA, IgD, IgE, IgG, IgM
IgG
2 heavy chains, 2
light chains
Papain cleavage
gets Fab and Fc
Heavy chain has 5
types a,d,e,g,m for
IgA, IgD, IgE, IgG,
IgM, respectively
Light chain has
types k and l
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b structure:
Immunoglobulin fold
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MHC I
Phagocytosis of an antibody-bound virus by a macrophage
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IgA: found principally in secretions such as saliva,
tears, milk; can be monomer, dimer, or trimer
IgM: first antibody produced by B cells; major
antibody in the early stages of a primary immune
response
IgG: major antibody in secondary immune
response
IgE: allergic response; interact with basophil(呈鹼
性染色顆粒的白血球細胞 ) and histaminesecreting cells (mast cell肥大細胞)
When antigen is bound by IgE, Fc of IgE binds to
the Fc receptor of basophil or mast cell and
induces them to secret histamine that causes
dilation and increased permeability of blood
vessels
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Antibodies bind tightly and
specifically to antigen
Kd ~ 10-10 M
Binding specificity is determined by the residues
in the variable domains of the heavy and light
chains
Conformational change in antibody during binding
antigen ----Induced fit
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Polyclonal and monoclonal antibodies
Inject protein to an animal, obtained
polycolonal antibodies are a mixture of
antibodies that recognize different part of
the protein. They are synthesized by many
different B cells.
Monoclonal antibodies are synthesized by a
population of identical B cells (a clone).
They recognize the same epitope.
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ELISA (enzyme-linked immunosorbent assay)
Rapid screening and quantification of the
presence of an antigen in a sample
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Test HSV antibody in blood
1. coat HSV antigen to well
2. Add blood sample (anti-HSV antibody)
3. Add anti-human IgG (linked with horseradish peroxidase)
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4. Add substrate: yellow (positive signal)
Immunoblot assay
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Protein interaction modulated by
chemical energy: actin, myosin
Motor proteins:
Kinesin and dynein move along
microtubules in cells, pulling along
organelles or reorganizing chromosomes
during division.
Helicase and polymerase move along DNA
Myosin and actin in muscle; actin and
myosin make 80 % of protein mass of
muscle
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Myosin (Mr 540,000) has 6 subunits: 2 heavy
chains (Mr 220,000) and 4 light chains (Mr 20,000)
Heavy chain: a-helix at C-terminal, forms lefthanded coiled coil with another heavy chain
N-terminal of each heavy chain has ATPase activity
and associates with 2 light chains
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Myosin forms “thick filament” in muscle, one
contractile unit has several hundred myosin
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Right handed
Monomeric actin is called G-actin (globular actin, Mr
42,000)
G-actin will associate to form a long polymer called Factin (filamentous actin)
“Thin filament” in muscle consists of F-actin, troponin and
tropomyosin.
In the formation of F-actin, each G-actin binds ATP, then
hydrolyzes it to ADP during association step. So in F-actin,
each actin molecule is complexed to ADP
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Each actin molecule in the thin filament can bind
tightly and specifically to one myosin head group
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Each muscle fiber is a single, very large, multinucleated cell
20 to 100 mm in diameter
It is formed from many cells fused together and often
spanning the length of the muscle.
Each fiber has about 1000 myofibrils, consisting vast amount
of regularly arrayed thick and thin filaments
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When muscle contracts, the I bands
narrow and the Z disks come closer
relaxed
contracted
Z disk is the anchor to which
the thin filaments are attached
M line is the high
electron density region
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The thick and thin filaments are interleaved
(交錯排列)
Each thick filament is surrounded by six
thin filaments
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There are other proteins involved in the sarcomere.
The actin filaments assembled with a-actinin, desmin,
vimentin, nebulin(~7000 residues) attach to the Z disk
Except myosin, M line has paramyosin, C-protein, Mprotein
Titin (26926 residues, the largest single polypeptide chain)
links the thick filament to the Z disk. It regulates the
length of sarcomere and prevents overtension of the
muscle.
Nebulin and titin ----molecular ruler ----regulate the length
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of thin and thick filaments
reference
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Myosin thick filaments slide
along actin thin filaments and
move toward Z disk
Each cycle generates 3 to 4 pN
of force, moves 5 to 10 nm
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How to regulate the contraction?
Tropomyosin forms a helix around the actin helix
covering all the myosin binding sites.
Troponin is Ca2+ -binding protein. A nerve impulse
causes Ca2+ release from sarcoplasmic reticulum.
Ca2+ binds to troponin and causes conformational
change in tropomyosin-troponin complex, exposing
the myosin binding sites on the thin filaments
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