Lecture 4

Protein structure and function

Sample questionsSample questions

Give brief definitions or unique descriptions of the following terms:

• (a) oligopeptide/peptide

• (b) quaternary structure

• (c) zwitterion

A short polymer of amino acids, usually up to ~50 residues

Spatial arrangement of subunits in a multi subunit protein

Molecule contain both a positive and a negative charged functional group

Protein structure review• Primary structure refers to the amino acid sequence of a protein (inc

luding the covalent bonds that link amino acids).

• Secondary structure refers to stable arrangements of amino acid residues giving rise to recurring structural patterns.

• Supersecondary structure (or motif) refers to particularly stable arrangements of several elements of secondary structure.

• Tertiary structure includes all aspects of the three-dimensional struct

ure of a protein

• Quaternary refers to the three-dimensional arrangement of two or more polypeptide subunits.

Sample questionsSample questions

• The Alpha Helix Is a Coiled Structure Stabilized by ______ Hydrogen Bonds

• Beta Sheet Is Stabilized by ______Hydrogen Bonds

A. Intrachain B. Interchain

A

B

Dihedral angles of protein

• To avoid atomic collisions, only certain angles are allowed.

• The angles in proteins are restricted to certain values

When phi angle is zero, no matter what the psi angle is, there are atomic collisions between O1 and C2.

Ramachandran plot

All you need to know is:

• Ramachandran plot represents sterically allowed conformations of the protein backbone

• We can predict the protein secondary structure from the phi and psi angle.

Ramachandran plot

• Can be used to check the quality of a protein structure

Amino Acid Ionization State as a Function of pH

This equation tell us when there is equal amount of A- and HA, pH = pKa

Henderson-Hasselbalch Equation

Log (1) = 0

Titration

• If sodium hydroxide is added to water.

• When the pH = pKa, there are equivalent amounts of the conjugate

acid and base. 

pHvalue

NaOH volume

HA exhausted

[HA] = [A-]

A- showed up

Titration of glycine

• The pKa of the α-COOH group is 2.4, whereas that of the α-NH3 + group is 9.8.

Titration Curve for Alanine

pI (isoelectric point) = the pH at which the number of positive and

negative charges on a population of molecules is equal (i.e. no net charge).

pK1 carboxylic acid = 2

pK2 amino group = 10

pI = (pK1+ pK2)/2

Sample questionsSample questions

• At a pH >pI of a given protein, that protein becomes ______, at the pH<pI of that same protein, it becomes _______.

• A. negatively charged (an anion)

• B. positively charged (a cation)

A

B

The isoelectric point (pI) is the pH at which a particular molecule or surface carries no net electrical charge.

The Purification of Proteins Is an Essential First Step in Understanding Their Function

• “Never waste pure thoughts on an impure protein”.• Proteins Must Be Released from the Cell to Be

Purified In the first step, a homogenate is formed by disrupting the cell membrane, and the mixture is fractionated by centrifugation.

• Proteins Can Be Purified According to Solubility, Size, Charge, and Binding Affinity

Salting Out (by solubility)• Most proteins are less soluble at high salt concentrations, an effect

called salting out. • The salt concentration at which a protein precipitates differs from

one protein to another. Hence, salting out can be used to fractionate proteins.

• Dialysis can be used to remove the salt if necessary.

Gel-Filtration Chromatography(by size)

• The sample is applied to the top of a column consisting of porous beads

• Large molecules flow more rapidly through this column and emerge first

• Small molecules can enter these beads and exit slowly.

• The negatively charged SDS (sodium dodecyl sulfate)-protein complexes migrate in the direction of the anode, at the bottom of the gel.

• The polyacrylamide gel separates proteins according to size, with the smallest moving most rapidly.

Polyacrylamide Gel Electrophoresis (by size)

Ion-Exchange Chromatography(by charge)

• Positively charged proteins (cationic proteins) can be separated on negatively charged columns.

• Conversely, negatively charged proteins (anionic proteins) can be separated by chromatography on positively charged columns.

• Proteins that have a low density of net positive charge will tend to emerge first, followed by those having a higher charge density.

Affinity Chromatography(by Binding Affinity)

• For example, the plant protein concanavalin A can be purified by passing a crude extract through a column of beads containing covalently attached glucose residues.

• Affinity chromatography is a powerful means of isolating transcription factors, proteins that regulate gene expression by binding to specific DNA sequences. A protein mixture is percolated through a column containing specific DNA sequences attached to a matrix; proteins with a high affinity for the sequence will bind and be retained.

Quantification of the purification protocol by Electrophoretic Analysis

Sample questionsSample questions• Consider the following ion exchange chromatography test at pH 6.0. A mixtu

re of proteins were applied to the column in a low [NaCl] buffer. Proteins retained by the cation exchange column were eluted by gradually increasing the NaCl concentration within the buffer. What can be concluded regarding the pI and relative charge of the protein(s) in each chromatogram peak?

Short summary

• Proteins can be purified according to solubility, size, charge, and binding affinity

• Proteins have an UV absorption peak at 280 nm

Hemoglobin, a molecule to breathe with

• Blood can carry very little oxygen in solution.

• Hemoglobin is required to carry oxygen around.

• Hemoglobin is found in red blood cells

• Each red blood cell can carry about one million molecules of oxygen

• Hemoglobin is 97% saturated when it leaves the lungs

• Under resting conditions is it about 75% saturated when it returns.

• The main function of red blood cell• Transfer of O2 from lungs to tissue• Transfer of CO2 from tissue to lungs

• To accomplish this function red cells has hemoglobin (Hb)

• Each red blood cell has 640 million molecules of Hb

Introduction

The α2β2 Tetramer of Human Hemoglobin

• The structure of the two identical α subunits (red) is similar to but not identical with that of the two identical β subunits

• The molecule contains four heme groups• Bind up to four oxygen molecules.

The iron-containing oxygen-transport metalloprotein in the red blood cells of all vertebrates

In mammals, the protein makes up about 96% of the red blood cells' dry content.

Hydrophobic interactions between subunits

Electrostatic interactions between subunits

Tertiary folding gives rise to at least 3 functionally important characteristics of the hemoglobin molecule

• 1- Polar or charged side chains tend to be directed to the outside surface of the subunit and, conversely, non-polar structures tend to be directed inwards. The effect of this is to make the surface of the molecule hydrophilic and the interior hydrophobic.

• 2- An open-toped cleft in the surface of the subunit known as heme pocket is created. This hydrophobic cleft protects the ferrous ion from oxidation.

• 3- The amino acids, which form the inter-subunit bonds responsible for maintaining the quaternary structure, and thus the function.

Structure of Heme•Heme contains:

conjugated system of double bonds → red colour

4 nitrogen (N) atoms

1 iron cation (Fe2+)

→ bound in the middle of

tetrapyrrole skelet by

coordination covalent bonds

• At the core of the molecule is porphyrin ring which holds an iron atom.

• An iron containing porphyrin is termed a heme.

• This iron atom is the site of oxygen binding.

• The name hemoglobin is the concatenation of heme and globin

Types of hemoglobin Adult Hb (Hb A)

•HbA1 (2 α and 2 β subunits) is the major form of Hb in adults and in children over 7 months.

•HbA2 (2 α, 2 δ) is a minor form of Hb in adults. It forms only 2 – 3% of a total Hb A.

Fetal Hb (Hb F) = 2 α and 2 γ subunits

- in fetus and newborn infants Hb F binds O2 at lower tension than Hb A → Hb F has a higher affinity to O2

•After birth, Hb F is replaced by Hb A during the first few months of life.

Hb S – in β-globin chain Glu is replaced by Val

= an abnormal Hb typical for sickle cell anemia

Fetal Hb vs. adult Hb

Pressure units:1 mmHg = 1 Torr1 mmHg = 133.22 Pa1 Pa = 0.0075 mmHg

Hb A Hb A2 Hb F

structure 22 22 22

Normal % 96-98 % 1.5-3.2 % 0.5-0.8 %

Adult hemoglobin

Hemoglobin

• Hemoglobin is a remarkable molecular machine that uses motion and small structural changes to regulate its action.

• Oxygen binding at the four heme sites in hemoglobin does not happen simultaneously.

• Once the first heme binds oxygen, it introduces small changes in the structure of the corresponding protein chain.

Hemoglobin

• These changes nudge the neighboring chains into a different shape, making them bind oxygen more easily.

• Thus, it is difficult to add the first oxygen molecule, but binding the second, third and fourth oxygen molecules gets progressively easier and easier.

• This provides a great advantage in hemoglobin function.

• When blood is in the lungs, where oxygen is plentiful, oxygen easily binds to the first subunit and then quickly fills up the remaining ones.

Hb must bind oxygen in lungs and release it in capillaries

Models for Allosteric Behavior

• Monod, Wyman, Changeux (MWC) Model: allosteric proteins can exist in two states: R (relaxed) and T (taut)

• In this model, all the subunits of an oligomer must be in the same state

• T state predominates in the absence of substrate S (Oxygen in our case)

• S binds much tighter to R than to T

More about MWC

• Cooperativity is achieved because S binding increases the population of R, which increases the sites available to S

• Ligands such as S are positive homotropic effectors

• Molecules that influence the binding of something other than themselves are heterotropic effectors

• Adjacent subunits' affinity for oxygen increases after the first oxygen binding.

• This is called positive cooperativity

Hemoglobin

• When oxygen level is low, hemoglobin releases its bound oxygen. As soon as the first oxygen molecule drops off, the protein starts changing its shape.

• This prompts the remaining three oxygens to be quickly released.

• In this way, hemoglobin picks up the largest possible load of oxygen in the lungs, and delivers all of it where and when needed.

Process of O2 binding to Hb

•Hb can exist in 2 different forms: T-form and R-form.

•T-form (T = tense, or taut) has a much lower oxygen affinity than the R-form. The subunits of Hb are held together by electrostatic interactions. The binding of the first O2 molecule to subunit of the T-form leads to a local conformational change that weakens the association between the subunits → R-form („relaxed“) of Hb.

•Increasing of oxygen partial pressure causes the conversion of T-form to R-form. • T R

• Hb + ↑pO2 HbO2

• When a first oxygen binds to Fe in heme of Hb, the heme Fe is drawn into the plane of the porphyrin ring

• This initiates a series of conformational changes that are transmitted to adjacent subunits

Heme Binding Site After O2 Binds

•O2 binds at distal side of Heme

•Fe2+ moves into porphyrin plane

Heme Prosthetic Group

Prosthetic Group = non-polypeptide unit of a protein that can function without the protein

Hemoproteins• Hemoglobin (Hb)• Myoglobin (Mb)• Cytochromes • Catalases (decomposition of

2 H2O2 to 2 H2O and O2)

• Peroxidases

Myoglobin

Mb is a monomeric heme protein • Mb polypeptide "cradles" the heme group

• Fe in Mb is Fe2+ - ferrous iron - the form that binds oxygen

• Oxidation of Fe yields 3+ charge - ferric iron -methemyoglobin does not bind oxygen

• Myoglobin provides muscle tissue with an oxygen reserve AND facilitates oxygen movement in muscle

One of the first proteins characterized by X-Ray Crystallography (Kendrew, 1959)

Myoglobin and hemoglobin monomer are structurally similar

Compare hemoglobin and myoglobin

A classic example of allostery

• Hemoglobin and myoglobin are oxygen transport and storage proteins

• Compare the oxygen binding curves for hemoglobin and myoglobin

• Myoglobin is monomeric; hemoglobin is tetrameric

• Mb: 153 aa, 17,200 MW

• Hb: two alphas of 141 residues, 2 betas of 146

-sigmoidal curve is shallow at low oxygen concentrations, low concentrations indicates that all molecules are in the T state-sigmoidal curve is steep when molecules increase at the R state and then it plateaus when the sites are filled

Binding of Oxygen by Hb

The Physiological Significance • Hb must be able to bind oxygen in the lungs

• Hb must be able to release oxygen in capillaries

• If Hb behaved like Mb, very little oxygen would be released in capillaries

• The sigmoid, cooperative oxygen binding curve of Hb makes this possible!

The Conformation Change

The secret of Mb and Hb!

• Oxygen binding changes the Mb conformation

• Without oxygen bound, Fe is out of heme plane

• Oxygen binding pulls the Fe into the heme plane

• Fe pulls its His F8 ligand along with it

• The F helix moves when oxygen binds

• Total movement of Fe is 0.029 nm - 0.29 A

• This change means little to Mb, but lots to Hb!

The energy in the formation of the Fe-O2 bond formation drives the T R transition.

Hemoglobins O2 -binding Cooperativity derives from the T R Conformational shift.

•The Fe of any subunit cannot move into its heme plane without the reorientation of its proximal His so as to prevent this residue from bumping into the porphyrin ring.

•The proximal His is so tightly packed by its surrounding groups that it can not reorient unless this movement is accompanied by the previously described translation of the F helix across the heme plane.

•The F helix translation is only possible in concert with the quaternary shift.

Molecular Shift at the Heme Group after Oxygen Binding

Blue: deoxyRed: oxy

Fe at the Heme Plane after Oxygen Binding

A Quaternary Structure Change

One alpha-beta pair moves relative to the other by 15 degrees upon oxygen binding

This massive change is induced by movement of Fe when oxygen binds

Conformational Changes• The movement of the histidine causes the movemen

t of the F-helix. This causes a corresponding rearrangement of the other helices in the protein subunit.

• The conformational change of the subunit causes it to move away from its partner in the pair. Movement of one subunit in the pair causes a corresponding conformational adjustment in the paired subunit, enhancing the ability of the latter to bind oxygen.

Hemoglobin: Axis of Symmetry Channel View

2 22

11

2

11

The Oxy form (red) forms a more condensed channel along the interface as compared to the Deoxy form (blue)

The reactivity of the heme group is different in the presence or absence of the polypeptide

• CO binds 25,000 times as strongly as O2 in the isolated heme, but only 200 times as strongly as O2 in myoglobin or hemoglobin

Heme

O2

Histidine

• CO Binding in Myoglobin and Hemoglobin– CO is a poison because it d

isplaces O2

– CO prefers linear coordination, O2 bent

– Distal His forces bent coordination of CO

– Let O2 compete with CO• CO produced in body takes

1% Hb • Without Distal His, CO > 99

% Hb

The reactivity of the heme group is different in the presence or absence of the p

olypeptide 11

25,000

N Fe OO

N Fe C O

200N Fe C

O

ISOLATED HEME

ISOLATED HEME

HEME WITH POLYPEPTIDE ENVIRONMENT

Hb-oxygen dissociation Hb-oxygen dissociation curvecurve

Hb-oxygen dissociation Hb-oxygen dissociation curvecurve

The normal position of curve The normal position of curve depends ondepends on

Concentration of 2,3-DPGConcentration of 2,3-DPG HH++ ion concentration (pH) ion concentration (pH) COCO22 in red blood cells in red blood cells Structure of HbStructure of Hb

When oxygen affinity is increased, the dissociation curve is shifted Leftward, and the value is reduced.Conversely, with decreased oxygen affinity, the curve is shifted to the right.

Hb-oxygen dissociation Hb-oxygen dissociation curvecurve

Right shift (easy oxygen delivery)Right shift (easy oxygen delivery)

High 2,3-DPGHigh 2,3-DPG High HHigh H++

HbSHbS

Left shift (give up oxygen less readily)Left shift (give up oxygen less readily) Low 2,3-DPGLow 2,3-DPG HbFHbF

Effects of carbon dioxide. Carbon dioxide affects the curve in two ways: first, it influences intracellular pH (the Bohr effect), second, CO2 accumulation causes carbamino compounds to be generated through chemical interactions. Low levels of carbamino compounds have the effect of shifting the curve to the right, while higher levels cause a leftward shift.

The Bohr EffectCompetition between oxygen and H+

• Discovered by Christian Bohr

• Binding of protons diminishes oxygen binding

• Binding of oxygen diminishes proton binding

• Important physiological significance

• Hemoglobin releases H+ when it binds O2

• Hemoglobin binds H+ when it releases O2

• HbO2 + H+ HbH+ + O2

Bohr Effect II

Carbon dioxide diminishes oxygen binding

• Hydration of CO2 in tissues leads to proton production

• These protons are taken up by Hb as oxygen dissociates

• The reverse occurs in the lungs

Carbon Dioxide (CO2)

• Transport of carbon dioxide by red cells, unlike that of oxygen, does not

occur by direct binding to heme.

• In aqueous solutions, carbon dioxide undergoes a pair of reactions:

1. CO2 + H2O H2CO3

2. H2CO3 H+ + HCO3

(CO2)• Carbon dioxide diffuses freely into the red cell where the

presence of the enzyme carbonic anhydrase facilitates reaction 1.

• The H+ liberated in reaction 2 is accepted by deoxygenated hemoglobin, a process facilitated by the Bohr effect.

• The bicarbonate formed in this sequence of reactions diffuses freely across the red cell membrane and a portion is exchanged with plasma Cl-, a phenomenon called the "chloride shift." the bicarbonate is carried in plasma to the lungs where ventilation keeps the pCO2 low, resulting in reversal of the above reactions and excretion of CO2 in the expired air.

• About 70% of tissue carbon dioxide is processed in this way. Of the remaining 30%, 5% is carried in simple solution and 25% is bound to the N-terminal amino groups of deoxygenated hemoglobin, forming carbaminohemoglobin.

Physiological Bohr Effect

Hb and the Bohr Effect (cont)• An equivalent amount of H+ released (about 0.

31 H+ per oxygen bound) is picked up by Hb as acid (H+) produced from tissue metabolism.

• At the lungs, when oxygen rebinds, the H+ released is converted to carbon dioxide and exhaled. About 40% of acid produced by the tissues is buffered in this way by Hb

Hemoglobin and the Bohr Effect

• The Bohr Effect is the direct result of the conformational changes that occur in Hb during oxygen binding.

• About 50% of the effect is caused by a change in pKa of His 146 (on beta subunit).

• In the T-state, the His is H-bonded to Asp 94. When Hb shifts from the T to R conformation, the H-bond to Asp 94 is disrupted, allowing a proton on the His to readily dissociate.

• The remainder of the effect results from similar pKa changes of other ionizable groups as a consequence of the T to R transition. This is the O2 bind/H+ release portion of the effect

Bohr Effect Summary

• The change in oxygen affinity with pH is known as the Bohr effect. • Hemoglobin oxygen affinity is reduced as the

acidity increases. • Since the tissues are relatively rich in carbon

dioxide, the pH is lower than in arterial blood; therefore, the Bohr effect facilitates transfer of oxygen.

• The Bohr effect is a manifestation of the acid-base equilibrium of hemoglobin.

2,3-diphosphoglycerate (2,3-DPG)

• This compound is synthesized from glycolytic intermediates.

• In the erythrocyte, 2-3-DPG constitutes the predominant phosphorylated compound, accounting for about two thirds of the red cell phosphorus.

• The proportion of 1,3-DPG pathway appears to be related largely to cellular ADP and ATP levels; when ATP falls and ADP rises, a greater proportion of 1,3-DPG is converted through the ATP-producing step.

• This mechanism serves to assure a supply of ATP to meet cellular needs.

• In the deoxygenated state, hemoglobin A can bind 2,3-DPG in a molar ratio of 1:1, a reaction leading to reduced oxygen affinity and improved oxygen delivery to tissues.

Physiological Function of DPG

• The presence of DPG in the erythrocyte creates a dynamic competition for oxygen binding to deoxy-Hb. DPG bound to deoxy-Hb reversibly stabilizes the deoxy conformation.

• As [DPG] increases, the greater the percentage of oxygen that will be released by Hb at the peripheral tissues. This process occurs naturally due to the differences in oxygen pressures between the lung and periphery, plus the presence of DPG further promotes this oxygen release.

• This is one mechanism to increase oxygen delivery to tissues during exercise and/or at high altitudes

2,3-BisphosphoglycerateAn Allosteric Effector of Hemoglobin

• In the absence of 2,3-BPG, oxygen binding to Hb follows a rectangular hyperbola!

• The sigmoid binding curve is only observed in the presence of 2,3-BPG

• Since 2,3-BPG binds at a site distant from the Fe where oxygen binds, it is called an allosteric effector

2,3-BPG and Hb

• Where does 2,3-BPG bind? – "Inside"

– in the central cavity

• What is special about 2,3-BPG? – Negative charges interact with 2 Lys, 4 His,

2 N-termini

• Fetal Hb - lower affinity for 2,3-BPG, higher affinity for oxygen, so it can get oxygen from mother

2,3 Diphosphoglycerate (DPG)

2,3 Diphosphoglycerate (DPG) (or called 1,3 bisphosphoglycerate (BPG)) binds tightly to the channel interface of deoxyHb, a bindingstabilized by interactions with positively charged sidechains. DPG has a poor binding affinity for oxyHb due to the compacted channel

• When oxygen is unloaded by the hemoglobin molecule and 2,3 DPG is bound, the molecule undergoes a conformational change becoming what is known as the ""Tense, Taut" or "T" form.

• The resultant molecule has a lower affinity for oxygen.

• As the partial pressure of oxygen increases, the 2,3, DPG is expelled, and the hemoglobin resumes its original state, known as the "relaxed" or "R" form, this form having a higher oxygen affinity.

• These conformational changes are known as "respiratory movement".

• The increased oxygen affinity of fetal hemoglobin appears to be related to its lessened ability to bind 2,3-DPG.

• The increased oxygen affinity of stored blood is accounted for by reduced levels of 2,3-DPG.

• Changes in 2,3-DPG levels play an important role in adaptation to hypoxia. In a number of situations associated with hypoxemia, 2,3-DPG levels in red cells increase, oxygen affinity is reduced, and delivery of oxygen to tissues is facilitated.

• Such situations include abrupt exposure to high altitude, anoxia due to pulmonary or cardiac disease, blood loss, and anemia.

• Increased 2,3-DPG also plays a role in adaptation to exercise. However, the compound is not essential to life; an individual who lacked the enzymes necessary for 2,3-DPG synthesis was perfectly well except for mild polycythemia

Hb and Carbon Dioxide Transport

• Some of the dissolved CO2 in the blood reacts with amino groups on Hb (and other proteins). This reaction occurs with N-terminal amino groups in the NH2 form;

• At blood pH, the side chain groups are in the NH3

+ form and will not react. About 15% of tissue-produced CO2 is bound by protein in this way (termed carbamino-CO2)

Interactions between DPG, Carbamino-CO2 and Bohr Effect

• The carbamino-CO2 can be bound to some of the same groups involved in DPG binding, thus diminishing DPG binding; the reverse can occur, DPG can block CO2

• CO2 also binds to some of the groups involved in the Bohr effect; thus bound CO2 can diminish the H+ buffering capacity of hemoglobin (again the reverse effect can occur, bound H+ blocking CO2

Methemoglobinemia

• In order to bind oxygen reversibly, the iron in the heme moiety of hemoglobin must be maintained in the reduced (ferrous) state despite exposure to a variety of endogenous and exogenous oxidizing agents.

• The red cell maintains several metabolic pathways to prevent the action of these oxidizing agents and to reduce the hemoglobin iron if it becomes oxidized. Under certain circumstances, these mechanisms fail and hemoglobin becomes nonfunctional.

(mět'hē-mə-glō'bə-nē'mē-ə)

Methemoglobinemia

• At times, hemolytic anemia supervenes as well. These abnormalities are particularly likely to occur (1) if the red cell is exposed to certain oxidant drugs

or toxins (2) if the intrinsic protective mechanisms of the cell are defective or (3) if there are genetic abnormalities of the hemoglobin molecule affecting globin stability or the heme crevice.

Sickle Cell Anemia

• The most common form of sickle cell anemia is caused by a single amino acid substitution of valine for glutamate at position 6 on the -subunit of hemoglobin.

• This defect is an autosomal (non-sex chromosome) recessive inherited disease, meaning both parents must be heterozygous carriers to produce a homozygous child.

• Even heterozygous carriers can experience sickle cell symptoms after vigorous exercise or unpressurized travel at high altitudes.

Sickle Cell Anemia (cont)

• The Glu to Val mutation in sickle cell hemoglobin (termed Hb-S) reduces the solubility of deoxyhemoglobin and allows formation of fibrous polymeric filaments of deoxyhemoglobin that precipitate in the red blood cells.

• This precipitation leads to an ultrastuctural deformity of the red blood cell, the "sickle" shape, which gives these cells a tendency to get hung up and accumulate in the narrow capillaries (thus leading to the associated peripheral pain and many other complications).

Subunit interactionsthat promote polymerization ofdeoxy-HbS

Sickle Cell Anemia (cont)• Upon exposure of oxygen at the lungs, the HbS filament

s immediately dissolve. • Thus, situations that slow down flow of blood through the

capillaries (normally 0.5 to 2 secs) can lead to the periodic sickle cell anemia “crises”. Conditions like oxygen deprivation (such as high altitudes or strenuous exercise) and dehydration can contribute to slower capillary passage of erythrocytes. The longer times allow deoxy-HbS polymerization to occur, and thus further contribute to the blockage of the affected area.

Other Types of Hemoglobin Mutations (Examples)

• Higher O2 affinity, 143 His to Gln; 2,3DPG binding decreased

• Lower O2 affinity, 102 Asn to Thr; T-form stabilized

• Methemoglobinemias, 3 His to Tyr; increased stability for Fe3+ caused by Tyr residue -helix disruptors, 3 His to Pro; Helix E disrupted, heme pocket opened, MetHb forms

• Decreased stability of Hb, 43 Phe to Val; heme pocket destabilized

Sickle Cell Anemia - Structure/Function Concepts

• Hb-S polymerization illustrates how one amino acid change from a charged to non-polar residue can lead to powerful, cumulative hydrophobic interactions.

• These are the same forces that hold the Hb monomer cores together, while the salt bridges and hydrogen bonds stabilize the surface interactions between subunits.

• Loss of the charged surface Glu allows more stable hydrophobic interactions to form in the deoxy-Hb conformation.

SummarySummary of Hemoglobin of Hemoglobin

Oxygen delivery to the tissuesOxygen delivery to the tissues Reaction of Hb & oxygenReaction of Hb & oxygen

Oxygenation not oxidationOxygenation not oxidation One Hb can bind to four OOne Hb can bind to four O22 molecules molecules chain move closer when oxygenatedchain move closer when oxygenated When oxygenated 2,3-DPG is pushed outWhen oxygenated 2,3-DPG is pushed out chains are pulled apart when Ochains are pulled apart when O22 is unloaded, is unloaded,

permitting entry of 2,3-DPG resulting in lower affinity permitting entry of 2,3-DPG resulting in lower affinity of Oof O22

Short Summary

• Cooperativity of oxygen binding to hemoglobin

• Modulators of oxygen binding to hemoglobin and the Bohr Effect

• Blood pH, and partial pressure of carbon dioxide influence hemoglobin saturation by altering hemoglobin's three-dimensional structure, thus changing its affinity for oxygen.

Sample questionsSample questions

• What is the prosthetic group that hemoglobin and myoglobin's oxygen binding ability depend on?

• Define cooperativity relative to binding oxygen

• What are the two states of the hemoglobin quaternary structure? And what are their characteristics?

Heme

binding of oxygen at one site increases chances of binding oxygen at the other sites; loss of anoxygen at one site increases the chances of losing oxygen at the other sites

T state = taut (deoxy form)R state = relaxed (oxygenated form)

Derivatives of hemoglobin Oxyhemoglobin (oxyHb) = Hb with O2

Deoxyhemoglobin (deoxyHb) = Hb without O2

Methemoglobin (metHb) contains Fe3+ instead of Fe2+ in heme groups

Carbonylhemoglobin (HbCO) – CO binds to Fe2+ in heme in case of CO poisoning or smoking. CO has 200x higher affinity to Fe2+ than O2.

Carbaminohemoglobin (HbCO2) - CO2 is non-covalently bound to globin chain of Hb. HbCO2 transports CO2 in blood (about 23%).