Chem 153A Week 3
Transcript of Chem 153A Week 3
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Chapter 8
Three Dimensional Structures ofProteins
Instructor: Rashid Syed
Textbook: Biochemistry (4th
Edition ) by Donald Voet Judith G. Voet
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Protein Structure
4 levels of protein structure
Primary structure: aa sequence
Secondary structure: regular chainorganization pattern
Tertiary structure: 3D complex folding Quarternary structure: association
between polypeptides
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Primary Structure Amino acid sequence determines primary structure
Unique for each protein; innumerable possibilities
Gene sequence determines aa sequence
Each aa is called a residue; numbering (&synthesis) always from NH 2 end toward COOHend
Amino acids covalently attached to each other byan amide linkage called as a peptide bond.
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Peptide Bond
Peptide bonds are rigid (no free rotation around it) and planar (2 -C and -O=C-N-H- in one plane)
Partial double bond character due to resonance structures of peptide bond (bond length is 1.32 A o instead of 1.49 A o
(single) or 1.27 Ao
(double) Partial sharing of 2 electron pairs between C and N of
peptide bond
Due to steric hindrance, all peptide bonds in proteins are intrans configuration
The 2 bonds around the -carbon have freedom of rotationmaking proteins flexible to bend and fold
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The Peptide Bond is Planar
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Partial Double Bond
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Trans Configuration
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Secondary Structure
Secondary structure is the initial folding pattern(periodic repeats) of the linear polypeptide.
Secondary structure refers to relativearrangement of adjacent amino acids
3 main types of secondary structure: -helix, -
sheet and bend/loop Secondary structures are stabilized by hydrogen
bonds
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The -helix
The -helix is right-handed or clock-wise coil of theaa chain around a central longitudinal axis. (for L-isoforms left-handed helix is not viable due to sterichindrance)
The R groups of aa protrude outward from the helix Each turn has 3.6 aa residues and is 5.4 A o high
The helix is stabilized by H-bonds between N-H and
C=O groups of every 4th
amino acid -helices can wind around each other to form coiled
coils that are extremely stable and found in fibrousstructural proteins such as keratin, myosin (muscle
fibers) etc
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Right-handed -helix
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Right- and Left-handed helices
Structure
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Stability of -helix
Interactions between aa side chains can influence -helix
stability Glu-rich sequence destabilizes -helix at pH 7 because of
repulsion between charges Adjacent lys and / or arg are also unstable A 3-aa separation between unlike charges is ideal for
ionic interaction and imparts stability Bulky aa such as asn, cys, ser in close proximity do not
favor -helix (also because of their shape). Pro and gly do not participate in -helix. Pro has a rigid N-C bond since N is part of a ring. Poly-gly stretchesare more stable in an alternate coiled structure because of
greater flexibility
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Stability of -helix
The peptide bond has a small electricdipole because of it partial double bondcharacter
This imparts a partial + charge to the N-
terminus of the -helix ; and a negativecharge to the C-terminus
The overall helix dipole is enhanced bylack of H-bonding at the 4 terminal aa oneach end
Thus, - charged aa at N-end and +charged aa at C-end have stabilizing
effect
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-Pleated Sheet
Extended stretches of 5 or more aa are called -strands
-strands organized next to each other make -sheets
If adjacent strands are oriented in the same direction (N-endto C-end), it is a parallel -sheet, if adjacent strands run
opposite to each other, it is an antiparallel -sheet. There canalso be mixed -sheets
H-bonding pattern varies depending on type of sheet
R-groups protrude outward from the pleats. Interactions between R-groups influence stability. Gly and ala common
-sheets are usually twisted rather than flat
Fatty acid binding proteins are made almost entirely of -sheets
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-Turn / Bend / Loop
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Relative Probability for Amino Acids to occur inType of Secondary Structure
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Circular dichroism spectra to assess Secondary Structures
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Motifs
Also called supersecondary structure Motifs are very stable folding patterns of
secondary structure.
They are combinations of helices, sheets, bends etc. such motifs are seen repeatedly in many
different proteins. Many kinds of motifs are possible and are
given unique names such as -barrel, zincfingers, leucine zippers etc.
Multiple motifs make up protein domains
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Some Motifs
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-sheets
arranged as a barrel
Common in fattyacid binding
proteins
Motifs
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Tertiary Structure
3-D arrangement of amino acids Polypeptides undergo folding or bundling up.
Includes interactions between amino acids that are
far apart in primary structure The folding creates pockets and sites for binding
substrates, ligands and cofactors
Folding is such that non-polar residues are buriedinside, polar residues are exposed outwards toaqueous environment.
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Tertiary Structure
5 kinds of bonds stabilize tertiary structure: H- bonds, van der waals interactions, hydrophobicinteractions, ionic interactions and disulphidelinkages
In disulphide linkages, the SH groups of twoneighboring cysteines form a S-S- bond called as adisulphide linkage. It is a covalent bond, but readily
cleaved by reducing agents that supply the protons toform the SH groups again
Reducing agents include -mercaptoethanol and DTT
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Reduction of Disulfide Linkages
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Protein Denaturation
The information for every proteins 3-D foldedstructure is inherent in its sequence The naturally occuring pattern of protein folding is
called its native conformation
The unfolding or loss of protein native conformationis called protein denaturation
Changes in temperature, pH, salt concentration and presence of organic solvents or urea and detergents
cause protein denaturation Denaturation is reversible; removal of disrupting
agents cause renaturation. Most proteins renaturespontaneously, others require assistance.
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Co-operativity in Protein Denaturation
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Molecular Chaperones
Sometimes protein folding is facilitated by other proteinscalled molecular chaperones. Chaperones often associate with a polypeptide chain as it is
being synthesized and guide it to fold in the right
functionally active conformation. This usually needs some input of energy in the form of
ATP. Two classes of chaperones: Heat-shock proteins, Hsp70,
are found in cells stressed by heat. They bind to denatured proteins and prevent incorrect folding or aggregation Chaparonins: multi-polypeptide complexes that facilitate
folding by an elaborate mechanism
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Errors in Protein Folding
Various genetic disorders may be the result ofincorrect protein folding
The most common cause of cystic fibrosis is due tomisfolding of the CFTR protein resulting from a
single amino acid deletion in the primary structure.
Mad cow disease and other forms of spongiformencephalopathies, (also called as Prion diseases:
pr oteinaceous infection on ly) result from misfoldingof the Prion Protein (PrP) in the brain. Normallyfolded PrP is converted to misfolded PrP byinteraction with the latter.
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Structure of Myoglobin
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Structural Domains
Many proteins are organized into multiple domains Domains are compact globular units that are connected
by a flexible segment of the polypeptide lackingsecondary/tertiary structure
Domains usually have biological activity. Eachdomain contributes a specific function to the overall
protein
Different proteins may share similar domainstructures, eg: kinase-, cysteine-rich-, globin-domains
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Multi-Domain
Structure ofCa-ATPase
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Domains of c-Src protein
SH3 domain = ~ 60 AASH2 domain = ~100AASH3 domain= ~300 AA kinase domain
(phosphotransferase)
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Fibrous Proteins
Polypeptides chains are arranged in long sheetsor strands
Consist of one predominant secondary structure
Fibrous proteins are structural proteins: theirfunctions include providing shape and
mechanical strength Examples: collagen, -keratin, silk fibrion
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-Keratin
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Collagen
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Silk Fibroin
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Quaternary Structure
Association of more than one polypeptides
Each unit of this protein is called as a subunit andthe protein is an oligomeric protein
Subunits (monomers) can be identical or different The protein is homopolymeric or
heteropolymeric
Disulfide bonds usually stabilize the oligomer.Electrostatic / hydrophobic interactions may alsocontribute to quaternary structure stability.
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Quaternary Structure of Hemoglobin
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Methods for the 3-D Structure of a Protein
NMR spectroscopy and X-ray
crystallography are the two mostcommonly used methods todetermine the 3-D structures of
macromolecules.
Principles of NMR
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Principles of NMR
Nuclei act as small barmagnets and possess a net
magnetic moment
No Magnetic FieldIsotropic conditions
Application ofexternal Bo
B o
Precession aroundexternal Bo
Precession atthe Larmorfrequency
=
Very homogeneousMagnetic field
Principles of NMR
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One-dimensional NMR
Two-dimensional NMR
1H chemical shift
1 5 N c
h e m
i c a l s
h i f t
1H,15N HSQC
Three-dimensional NMR
Principles of NMR
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NMR Spectroscopy
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Wh t l d?
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Why are crystals used?
1. X-ray scattering from a single molecule would be incredibly weak and impossible to detect.
2. A crystal arranges a huge number of moleculesin the same orientation, so that scattered wavescan add up in phase and raise the signal to ameasurable level.
Principles of X ray crystallography
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Principles of X-ray crystallography
X Diff ti
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X-ray Diffraction
1.9
F i th
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Fourier theory
1. The diffraction pattern is related to theobject diffracting the waves through amathematical operation called the
Fourier transform.2. So the real image (electron electron
density) can be obtained by by takingthe Fourier transform as long as boththe amplitude and the phaseinformation is known
What is electron density?
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What is electron density?
A map of the distribution of electrons inthe molecule, i.e . an electron density map.
However, since the electrons are mostlytightly localized around the nuclei, theelectron density map is a pretty good
picture of the molecule.
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Crystallization Process
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Crystallization Process
1. For obtaining crystals, the molecules must
assemble into a periodic lattice.2. Start with a solution of the protein with a fairly high
concentration (2-50 mg/ml) and add reagents thatreduce the solubility close to spontaneousprecipitation (called superstaturated state).
3. With slowing further concentration, and underconditions suitable for the formation of a few
nucleation sites, crystalsmay start to grow.4. Many conditions are generally tested usually by
initial screening, then followed by a systematicoptimization of conditions.
Phase Diagram for Crystallization Experiment
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Phase Diagram for Crystallization Experiment
State A: Protein stays undersaturated and no crystals growState B: Protein crystallizes and the concentration of protein
in solution drops to saturationState C: Protein precipitates, but crystals may still grow
Crystallization Methods
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Crystallization Methods
1. Batch Method2. Vapour Diffusion Method
i) Hanging Dropii) Sitting Dropiii) Sandwich Drop
3. Dialysis Method
Batch Method
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Batch Method
The precipitant and protein are mixed directly
This can be done in a glass vial or under oil
Vapour Diffusion:Hanging Drop Method
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Vapour Diffusion:Hanging Drop Method
Vapour Diffusion: Sitt ing Drop Method
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Vapour Diffusion: Sitt ing Drop Method
Dialysis Method
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Dialysis Method
Protein is equilibrated against a larger volume ofprecipitant through a dialysis membrane.
X-ray crystallography requires a singlel h l
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crystal as the sample
But all you get is precipitate. Need to try more conditions.
X-ray crystallography requires a singlel h l
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crystal as the sample
A good quality crystal is grown for data collection!!!
Protein Structure determination
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Fe
HIS-313
HIS-374
ASP-315
TRP-389
Water
TYR-310
MET-299
TYR-303
Final Model of HIF-PH2 based on X-ray Crystallography
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Final Model of HIF PH2 based on X ray Crystallography
Ribbon Diagram of Crystal Structure of HIF-PH2
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Orderedamino acids188 to 403represented asa ribbondiagram
Mixed / structure with strand corefolded into a
jellyroll motif
Chapter 8
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p
Protein Function: Hemoglobin inMicrocosm
Instructor: Rashid Syed
Textbook: Biochemistry (4 th Edition ) by Donald Voet Judith G. Voet
Protein Function
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Protein Function
Reversible binding of ligands is essential Specificity of ligands and binding sites Ligand binding is often coupled to conformational changes, sometimes
quite dramatic ( Induced Fit )
In multisubunit proteins, conformational changes in one subunit can affectthe others ( Cooperativity )
Interactions can be regulated
Illustrated by :Structures of myoglobin and hemoglobinOxygen-binding curvesRegulation of O 2 binding
F ti f P t i
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Functions of Proteins Storage of ions and molecules
myoglobin, ferritin Transport of ions and molecules
hemoglobin, serotonin transporter Defense against pathogens
antibodies, cytokines Muscle contraction
actin, myosin Biological catalysis
chymotrypsin, lysozyme
Prosthetic Groups
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Prosthetic Groups
Some proteins have non-peptide components as part of the functional unit. These non-peptidecomponents are called as prosthetic groups.
Prosthetic groups are permanently associated withthe protein and are required for protein function
Proteins with prosthetic groups are called
complex or conjugated proteins. otherwise theyare simple proteins.
Ligands
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Ligands
A molecule that binds reversibly to a protein is calledits ligand. Ligands may be small peptides or non-
peptide molecules. Physiological ligands are usuallyendocrine, metabolic or environmental factors.
Ligand binding is a trigger for the activation of proteinfunction.
Ligand binding usually results in a conformationalchange in the proteins 3-D structure.
The ligand binding site on the protein iscomplementary to the ligand in size, shape and charge.
Ligand binding is specific.
Binding: Quantitative Description
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g Q p
Consider a process in which a ligand (L) binds reversibly to a site in the protein (P)
The kinetics of such a process is described by:the association rate constant k a
the dissociation rate constant k d
After some time, the process will reach the equilibrium
where the association and dissociation rates are equal The equilibrium composition is characterized by the the
equilibrium constant K a
+ k a
k d PLPL
d
aa
k
k K ==
]L[]P[
]PL[
]PL[]L[]P[ d a k k =
ak
K ==]PL[
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Think of this as % of ligand binding sites occupied.
100%
50%
% ligand binding sites occupied = = --------------------[L] n
[L] n + K d
How does Le Chatliers Principle apply?
d
ak ]L[]P[
Few ligands, Equilibrium not pushed
very far to right
Lots of ligands, equilibrium pushed very far to the right
Hyperbolic Curve for Ligand Binding
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Hyperbolic Curve for Ligand Binding
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Oxygen binding curve of Myoglobin
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Oxygen-binding curve of Myoglobin
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Structure of Heme
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St uctu e o e e
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Interactionsof aa withheme in
myoglobin
Structure of Myoglobin
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The primary structure is its sequence. This consists of 153
aa with M r = 16,700 Secondary structure: 8 alpha-helical regions designated as
A to H starting at the NH 2 terminus. No -sheets. The tertiary structure: folding of the 8 helices into a single
domain called the globin fold. Several bends and loops formed in the process; they are
labeled reflecting the helical segments they connect. Folding results in the formation of a pocket or box where
the heme group fits in. Hydrophobic aa are buried in the
interior, lining the heme pocket. The tertiary structure is stabilized mostly by hydrophobic
interactions. There are no disulphide bonds. Myoglobin is monomeric, there is no quarternary structure.
Structure of Myoglobin
Structure of Myoglobin
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Structure of Myoglobin
Hemoglobin subunits are structurallyl l b
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Similar to Myoglobin
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Hemoglobin Structure
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g Hemoglobin is an oligomeric protein made up of 2
dimers, a total of 4 polypeptide chains: 11 22. The (141 aa) and (146 aa) subunits are highly
homologous. Total M r of hemoglobin is 64,500
Each subunit consists of 7 ( ) or 8 ( ) alpha helices andseveral bends and loops folded into a single globindomain.
Each chain has one side where nonpolar groups areexposed rather than polar groups. These regions interact
such that the 4 chains are held together by hydrophobicinteractions. H-bonds and ionic interactions alsocontribute to quarternary structure.
Each subunit has a heme-binding pocket.
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Different forms of Hemoglobin
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Different forms of Hemoglobin
When hemoglobin is bound to O 2, it is calledoxyhemoglobin. This is the relaxed (R ) state.
The form with a vacant O 2 binding site is called
deoxy-hemoglobin and corresponds to the tense (T)state.
If iron is in the oxidized state as Fe +3, it is unable to bind O 2 and this form is called as methemoglobin
CO and NO have higher affinity for heme Fe +2 thanO2 and can displace O 2 from Hb, accounting for theirtoxicity.
Changes Induced by O 2 Binding
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g y 2 g
O 2 binding rearranges electrons within Fe making it morecompact so that it fits snugly within the plane of porphyrin.
Since Fe is bound to histidine of the globin domain, when Femoves, the entire subunit undergoes a conformational
change. This causes hemoglobin to transition from the tense (T) state
to the relaxed (R) state.
Inter-subunit interactions influence O 2 binding to all 4subunits resulting in cooperativity
O 2-binding triggers conformational change
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O 2 binding triggers conformational change
Inter-subunit interactions
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Inter subunit interactions
Two alpha and two beta subunits ( 11 22)
Strong interactions between unlike subunits
About 30 aa involved in inter-subunit interactions;
dimer remains intact after disruption with urea In deoxyhemoglobin, the C-terminal His and Asp of
-subunits interact with lys of -subunit. Also argand asp on C-terminus of one -subunit interact with
those of 2nd
-subunit. In oxyhemoglobin, the histidines have shifted to the
interior and can no longer form ion pairs
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T and R states of Hemoglobin
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g
Hemoglobin exists in two major conformationalstates: Relaxed (R ) and Tense (T)
R state has a higher affinity for O 2. In the absence of O
2, T state is more stable; when O
2
binds, R state is more stable, so hemoglobinundergoes a conformational change to the R state.
The structural change involves readjustment of
interactions between subunits. The 11 and 22 dimers rearrange and rotate
approximately 15 degrees with respect to each other
T-state to R-state Transition
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T-state to R-state Transition
O 2-binding kinetics
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4 subunits, so 4 O 2-binding sites
O 2 binding is cooperative meaning that each subsequent O 2 binds with a higher affinity than the previous one
Similarly, when one O 2 is dissociated, the other three willdissociate at a sequentially faster rate.
Due to positive cooperativity, a single molecule is very rarely partially oxygenated.
There is always a combination of oxygenated anddeoxygenated hemoglobin molecules. The percentage ofhemoglobin molecules that remain oxygenated is represented
by its oxygen saturation
O 2-binding curves show hemoglobin saturation as a functionof the partial pressure for O 2.
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SigmoidalCurve forO 2 binding
Oxygen Saturation Curve
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Oxygen Saturation Curve Saturation is maximum at very high O
2 pressure in the
lungs (pO 2 = ~ 100 torr).
As hemoglobin moves to peripheral organs and the O 2 pressure drops (pO 2 = ~20 torr), saturation also drops
allowing O 2 to be supplied to the tissues. Due to co-operative binding of O 2 to hemoglobin, its
oxygen saturation curve is sigmoid.
Such a curve ensures that at lower pO 2, smalldifferences in O 2 pressure result in big changes in O 2 saturation of hemoglobin. This facilitatesdissociation of O 2 in peripheral tissues.
O 2-transfer from Hemoglobin to Myoglobin
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2
The myoglobin curve shows us that only at extremelylow O 2 pressure, there is no binding of O 2 tomyoglobin.
At relatively low pO 2, the saturation shoots up and
there is almost complete saturation, meaning that allmolecules are associated with O 2.
At the pO 2 that myoglobin is fully saturated,hemoglobin is less than half saturated. This facilitatesthe transfer of O 2 from hemoglobin to myoglobin inthe muscle.
Effectors of O 2 binding
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Small molecules that influence the O 2-binding capacity
of hemoglobin are called as effectors (allostericregulation)
Positive or negative effectors; homotropic or heterotropiceffectors
Oxygen is a homotropic positive effector
Positive effectors shift the O 2-binding curve to the left,negative effectors shift the curve to the right
From a physiological view, negative effectors are beneficial since they increase the supply of oxygen to thetissues.
The Bohr Effect
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The regulation of O 2-binding to hemoglobin by H + and CO 2
is called the Bohr effect Both H + and CO 2 are negative effectors of O 2-binding.
Addition of a proton to His imidazole group at C-terminus of-subunit facilitates formation of salt bridge between His andAsp and stabilization of the T quaternary structure ofdeoxyhemoglobin.
CO 2 reduces O 2 affinity by reacting with terminal NH 2 toform negatively charged carbamate groups that form salt
bridges to stabilize deoxyhemoglobin.
Metabolically active tissues need more O 2; they generatemore CO 2 and H + which causes hemoglobin to release its O 2.
Effect of pH on Oxygen -Binding
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p yg g
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2,3-Bisphosphoglycerate
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, p p g y
2,3-Bisphosphoglycerate is a negative effector.
A single 2,3-BPG binds to a central pocket ofdeoxyhemoglobin and stabilizes it by interactingwith three positively charged aa of each -chain.
2,3-BPG is normally present in RBCs and shifts theO2-saturation curve to the right
Thus, 2,3-BPG favors oxygen dissociation and
therefore its supply to tissues In the event of hypoxia, the body adapts by
increasing the concentration of 2,3-BPG in the RBC
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Fetal Hemoglobin
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Fetal hemoglobin has 2 and 2 chains
The chain is 72% identical to the chain.
A His involved in binding to 2,3-BPG is replaced withSer. Thus, fetal Hb has two less + charge than adult Hb.
The binding affinity of fetal hemoglobin for 2,3-BPG issignificantly lower than that of adult hemoglobin
Thus, the O 2 saturation capacity of fetal hemoglobin is
greater than that of adult hemoglobin This allows for the transfer of maternal O 2 to the
developing fetus.
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Sickle-cell anemia is due toi i h l bi
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a mutation in hemoglobin
Glu6 Val in the chain of Hb The new Valine side chain can bind to a
different Hb molecule to form a strand
This sickles the red blood cells Untreated homozygous individuals
generally die in childhood Heterozygous individuals exhibit a
resistance to malaria
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