Post on 01-Jan-2016
Protein Classification• Simple – composed only of amino acid
residues
• Conjugated – contain prosthetic groups(metal ions, co-factors, lipids, carbohydrates)Example: Hemoglobin – Heme
Protein Classification• One polypeptide chain - monomeric protein• More than one - multimeric protein• Homomultimer - one kind of chain• Heteromultimer - two or more different
chains
(e.g. Hemoglobin is a heterotetramer. It has two alpha chains and two beta chains.)
Protein ClassificationFibrous –1) polypeptides arranged in long strands or
sheets2) water insoluble (lots of hydrophobic AA’s)3) strong but flexible4) Structural (keratin, collagen)
Globular –1) polypeptide chains folded into spherical or
globular form2) water soluble3) contain several types of secondary structure4) diverse functions (enzymes, regulatory
proteins)
Protein Function
• Catalysis – enzymes• Structural – keratin• Transport – hemoglobin• Trans-membrane transport – Na+/K+ ATPases• Toxins – rattle snake venom, ricin• Contractile function – actin, myosin• Hormones – insulin• Storage Proteins – seeds and eggs • Defensive proteins – antibodies
Globular Proteins Myoglobin/Hemoglobin
Hemeproteins: group of specialized proteins that contain
heme group as a tightly bound prosthetic group.
prosthetic group: is a non-protein compound that is
permanently associated with protein
The role of heme group is dependent on the environment
created by the three-dimensional structure of the protein.
e.g. heme in cytochrome electron carrier,
enzyme catalase active site
Myoglobin and hemoglobin Oxygen carrier
First protein structures determined
Oxygen carriers
Hemoglobin transport O2 from lungs to tissues
Myoglobin O2 storage protein
Myoglobin/Hemoglobin
Structure and function of Hemoglobin Mb and Hb subunits structurally
similar
myoglobin hemoglobin
•8 alpha-helices•Contain heme group•Mb monomeric protein•Hb heterotetramer (α22)
- Hb found exclusively in red blood cells-transport O2 from lungs to capillaries of tissues and transfer CO2 from tissues to lungs -composed of 4 polypeptides held together by non-covalent interaction-each subunit is similar to myoglobin and contains a heme group
The interior of the myoglobin is composed of NON-polar a.a. they packed together stabilized by hydrophobic interaction.Charged a.a are located at the surface.
-Myoglobin is O2 binding protein found in almost all mammals mainly in muscles and heart -its main function is to store O2 for periods where energy demands is high, it also increases the rate of transport of oxygen within the muscle cells.- Compact structure, 80% of its polypeptide chain is α-helix that labeled A to h that terminated by Proline or by -bends
porphyrine
protoporphyrine IX
Structure of HemeHeme is a complex of protoporphyrine IX (4 pyrrole rings linked by methene bridges) and ferrous iron (Fe+2) Ferrous ion has 6 coordination bonds: 4 with the N of pyrrole rings and 2 are perpendicular one with N of histidine and the other is with O2
Heme = Fe++ bound to tertapyrrole ring (protoporphyrin IX complex)
Heme non-covalently bound to globin proteins through His residue
O2 binds non-covalently to heme Fe++, stabilized through H-bonding with another His residue
Heme group in hydrophobic crevice of globin protein
Heme group
Heme = Fe++ bound to tertapyrrole ring (protoporphyrin IX complex)
Heme non-covalently bound to globin proteins through His residue
O2 binds non-covalently to heme Fe++, stabilized through H-bonding with another His residue
Heme group in hydrophobic crevice of globin protein
Distal histidine: stabilizes the binding of O2 to heme
Proximal histidine
Distal histidine: stabilizes the binding ofO2 to heme
Proximal histidine
Heme
Ferrous ion
Oxygen
Oxygen binding Oxygen binding to Heme groupto Heme group
Hb tetramer can be described as two identical dimers, (α)1 and (α)2The interaction between α and subunits is strong (hydrophobic, ionic and hydrogen interactions )The interaction (α)1 and (α)2 is weak interaction (primary hydrophobic). The two dimers can move with respect to each other two conformations according to the presence or absence of O2
T-form (Taut or tense): the deoxy form of Hb. The two α dimers interact through a network of ionic bonds and hydrogen bonds that constrain the movement of the dimer. This form is the low O2 affinity form of HbR-form (relaxed form): the binding of O2 causes rupture of some ionic bonds and H-bonds the polypeptide chains have more freedom of movement. The R-form is the high O2 affinity form of Hb.
Oxygen binding to MyoglobinOxygen binding to Myoglobin
Deg
ree o
f satu
rati
on
Concentration of Oxygen (Partial pressure)
Myglobin has one heme group bind only one oxygen molecule.Hemoglobin has 4 heme groups bind to 4 oxygen molecules O2 dissociation curve has hyperbolic shape
Oxygen dissociation curve
Myglobin has higher O2
affinity than of
Hemoglobin
P50 of Mb is about 1
mmHg and for Hb is 26
P50 is O2 Partial
pressure needed to half
saturation of the Mb of
Hb
Oxygen Oxygen transpotransport rt proteinproteinssEfficient O2
transport protein should bind to O2 at high partial pressure (loading in lung) and release it (low affinity) at low Partial pressure of (unloading in the tissue)
•Mb has hyperbolic O2 binding curve
•Mb binds O2 tightly. Releases at very low
pO2
•Hb has sigmoidal O2 binding curve
•Hb high affinity for O2 at high pO2 (lungs)
•Hb low affinity for O2
at low pO2 (tissues)
Oxygen Binding CurvesOxygen Binding Curves
tissues
lungs
Strong-binding
Weak-binding
Transition from weak to strong binding
OO22 Binding to Hb shows positive Binding to Hb shows positive cooperativitycooperativity
O2 Binding to Hb shows sigmoidal shape, low binding affinity at low con of Oxygen and high affinity at higher concooperative binding by the four subunit of Hb The binding of one O2 molecule at one heme group increases the oxygen affinity of the remaining heme groups in the same hemoglobin molecule. The affinity of hemoglobin for the last O2 bound is 300 times greater than its affinity for the first O2
O2 affinity increases as each O2 molecule bindsIncreased affinity due to conformation changeDeoxygenated form = T (tense) form = low affinityOxygenated form = R (relaxed) form = high affinity
Increasing affinity
for O2
Hb
Hemoglobin is efficient in delivering the O2 to the tissues from lung, myglobin which has hyperbolic O2-dissociation curve is unable to do that
Cooperative O2 Binding to HbCooperative O2 Binding to Hb
Myoglobin-Oxygen bindingMyoglobin-Oxygen binding
Allosteric InteractionsAllosteric Interactions
• Allosteric interaction occur when specific molecules bind a protein and modulates activity
• Allosteric modulators or allosteric effectors bind reversibly to site separate from functional binding or active site
• Modulation of activity occurs through change in protein conformation
• 2,3 bisphosphoglycerate (BPG), CO2 and
protons are allosteric effectors of Hb binding of
O2
CO2 + H2O H2CO3 HCO3 + H+
Most of the CO2 produced in metabolism is hydrated and transported
as bicarbonate ion. the hydration of CO2 by the zinc-dependent
enzyme carbonic anhydrase.
How is COHow is CO22 Exported? Exported?
Binding of Hemoglobin to CO2Binding of Hemoglobin to CO2
Some CO2 is carried as carbamate bound to the uncharged α-amino group
Carbon dioxide is transported in the form of a carbamate on the amino terminal residues of each of the polypeptide subunits.
Direct binding of CO2 to Hb stabilizes the T- form (deoxy) of Hb resulting in a decrease in its affinity for oxygen
The formation of a carbamate also results in release of a proton into solution indirectly induces the Bohr effect
Amino Terminus Carbamate on Amino Terminus
O
C
O
C C
R
H
O
ProteinH2N C C
R
H
O
ProteinHNC
O
O
H+
Bohr EffectBohr Effect Increased CO2 leads to
decreased pH CO2 + H2O <-> HCO3- + H+
At decreased pH several key a.a’s protonated, causes Hb to be converted to T-conformation (low affinity)HbO2 + H HbH + O2.
Deoxy form of Hb has higher affinity of H than O2
Protonation of some amino acids stabilizes the deoxyhemeglobin (T-form)
HCO3- combines with N-terminal α-amino group to form carbamate group.
Carbamation stabilizes T-conformation
Bisphosphoglycerate (BPG)Bisphosphoglycerate (BPG)
2,3-Bisphosphoglycerate is an important allosteric effecter of hemoglobin Binding of BPG to Hb causes low O2 affinity
One molecule binds at the interface of all four subunits, and makes contacts with the β
-subunits. BPG binds in the cavity between β-Hb subunits
Its binding stabilizes the deoxyhemoglobin state so stabilizes T-conformation. This promotes oxygen dissociation from oxyhemoglobin.
2,3-BPG concentration increases in response to chronic
hypoxia as in pulmonary obstruction or to high altitude or
chronic anemia. 2,3-BPG is present in erythrocytes at about 5 mM (at sea
level), At high altitudes it is present at 8 mM. Shifts the
curve to the right increase the O2 delivery to the tissue. At high altitudes, wherein the partial pressure of oxygen is
low, one would want hemoglobin to give up more of its
bound oxygen to the tissues Fetal Hb (α22) has low affinity for BPG, allows fetus to
compete for O2 with mother’s Hb (α2 β2) in placenta Role of 2,3-BPG in transfused blood
2,3-Bisphosphoglycerate2,3-Bisphosphoglycerate
Formation of methemoglobin:Formation of methemoglobin: • oxidation of the heme component of Mb and Hb into ferric (Fe+3)
state form metmyglobin and methmoglobin• The oxidized heme can't bind the O2• This oxidation can result from drugs or toxins or from inherited
defects • Occasional oxidation of heme is corrected by the enzyme NADH-
cytochrome b5 reductase that found in the red blood cell.• Methemoglobin binds strongly to CN (poison that inhibits the
cytochromal electron transport), so in the case of the cyanide poisoning amyle nitrite is taken which able to oxidize the heme group sequestering the CN
Carbon monoxide binding to HbCarbon monoxide binding to Hb• CO binds tightly to one or more of heme iron forming carbon
monoxyhemoglobin (HbCO) and hemoglobin is shifted to R-form causing the remaining heme with high O2 affinity shifts the O2-binding curve to the hyperbolic (left) inability of affected hemoglobin to deliver O2 to the tissue
Types of HemoglobinsTypes of HemoglobinsThere are 4 different types of hemoglobins known:
The most common is Hb A that form 90% of total Hb and consists of α2 β2Hb F (α22) less than 2%
Hb A2 (α22) 2-5%Hb A1C (α2 β2-glucose) 3-9 %
Fetal hemoglobin (Hb F): tetramer α22 • Hb F is major Hb in the fetus and newborn. During the last month of
pregnancy , it accounts for 60 % of the total Hb. • In the first few weeks of pregnancy embryonic Hb is synthesized Hb
Gower1 (ζ2ε2) after that the liver starts HbF synthesis. After the
development of the bone marrow the Hb A is synthesized at about the eighth month of the pregnancy and gradually replaces the Hb F
Binding of the 2,3-BPG to HbF (Binding of the 2,3-BPG to HbF (αα222)2) • HbF has higher affinity for O2 than dose HbA, bec it has lower binding
affinity to 2,3-BPG and this facilitates the transfer of O2 from maternal circulation across the placenta to the red blood cells of the fetus
2 globin chains (HbF) lack some positively charged amino acids found that found in the β globin (Hb A) reduce the 2,3BPG binding higher
affinity to O2
Hemoglobin A2 (Hb A2 (Hemoglobin A2 (Hb A2 (αα222))2))
• Hb A2 is a minor component of normal
adult hemoglobin, appear firstly about
12 week after the birth and can form
about 2% of the total Hb
Hemoglobin A1cHemoglobin A1c
• Under physiologic conditions HbA is
slowly and non-enzymatically
glycosylated
• The extent of glycosylation is
dependent on the plasma level of
particular hexoses
• The most abundant glycosylated Hb is
HbA1c which has glucose unit that
covalently linked to amino group of N-
terminal valines of the beta chain
• In the case of Diabetes mellitus, the
amount of HbA1c will increase
HemoglobinopathiesHemoglobinopathiesDefined as a family of disorders caused either by production of
structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin or rarely both
Sickle- cell anemia (HbS) Hemoglobin C disease (HbC)
Thalassemia
Sickle- cell anemia (Hemoglobin S disease “HbS”)Sickle- cell anemia (Hemoglobin S disease “HbS”)• a glutamate residue is replaced by valine residue in the β-chains.
This results in two fewer negative charges for the tetrameric structure.
• The substitution of a hydrophobic amino acid for a hydrophilic one makes the resulting molecule “sticky.” This is because a
hydrophobic patch has been created, which causes molecules to stick together at this point. This causes aggregation to occur in
deoxyhemoglobin. • Subsequent to strand formation, several strands can assemble to
form an insoluble fiber, which is what gives sickled cells there shape.
• People with sickle cell anemia suffer from repeated crises brought on by physical exertion.
HemoglobinopathiesHemoglobinopathies
Sickle- cell anemia (Hemoglobin S disease “HbS”)Sickle- cell anemia (Hemoglobin S disease “HbS”)
Hemoglobin C disease HbC is a hemoglobin variant having a single substitution in the sixth
position of the β-globin chain. In this case lysine is substituted. Patients have a relatively mild chronic hemolytic anemia and they
don't suffer from infractive crises Hemoglobin SC disease
in this disease some β-globin chains have sickle-cell mutation and other β-globin chains carry mutation found in HbC
Thalassemias Thalassemia is a hereditary hemolytic disease in which an imbalance
in the synthesis of globin chains occurs Normally the synthesis of α-chains and β-chains are coordinated so
that each α-globin has its β-globin in the thalassemia the synthesis of either α- or β-globin chain is
defectiveα-thalassemia: defect in the synthesis of the α-globin and there are 4
different levels of this type β-thalassemia: β-globin is decreased or absent, there are 2 different
level of this type
The EndThe End
2,3-bisphosphoglycerate binding to hemoglobin stabilizes the T state.
The negative charges on 2,3-bisphosphoglycerate interact with positive charges on hemoglobin (shown in blue)
Shown here is the R state of hemoglobin, to which oxygen has a greater affinity. Notice how the binding site for BPG collapses.
2,3-bisphosphoglycerate Binding to Hemoglobin
Sickle Cell Anemia• Sickle cell anemia was the first condition for which a genetic
mutation was correlated with a physiological response. This is a homozygous recessive condition, in which offspring must inherit both of the mutated genes in order to develop the disease fully.
• There are more than 300 different genetic variants of hemoglobin that are known.
• In the case of sickle cell disease, a valine residue is substituted for a glutamate residue in the b chains. This results in two fewer negative charges for the tetrameric structure.
• The substitution of a hydrophobic amino acid for a hydrophilic one makes the resulting molecule “sticky.” This is because a hydrophobic patch has been created, which causes molecules to stick together at this point. This causes aggregation to occur in deoxyhemoglobin.
• Subsequent to strand formation, several strands can assemble to form an insoluble fiber, which is what gives sickled cells there shape.
• People with sickle cell anemia suffer from repeated crises brought on by physical exertion. The hemoglobin content of their blood is about 1/2 of normal erythrocytes, and the sickled cells can block capillaries, causing severe pain.
Mutations in a- or b-globin genes can cause disease state
• Sickle cell anemia – E6 to V6
• Causes V6 to bind to hydrophobic pocket in deoxy-Hb
• Polymerizes to form long filaments
• Cause sickling of cells
• Sickle cell trait offers advantage against malaria
• Fragile sickle cells can not support parasite
Structure and function of HemoglobinStructure and function of Hemoglobin-found exclusively in red blood cells-transport O2 from lungs to capillaries of tissues and transfer CO2 from tissues to lungs -composed of 4 polypeptides held together by non-covalent interaction-each subunit is similar to myoglobin and contains a heme group
Oxygen Binding CurveOxygen Binding Curve
Oxygen Binding CurveOxygen Binding Curve