Chapter 4: PROTEINS: 3D STRUCTURE

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Chapter 4PROTEINS 3-D STRUCTURE
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How much does DNA sequencing tell us about the
three-dimensional structures and biological
functions of proteins?
QUESTION
3
What is the difference between genomics and
proteomics?
QUESTION
4
Central Dogma of Molecular Biology
DNA
Transcription
mRNA
Translation
Folded Protein
Amino Acids (20 standard)
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What doesnt the Human Genome Project tell us?
DNA
Transcription
mRNA
Translation
Folded Protein
Amino Acids
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Model Protein KERATIN

• Hair
• Porcupine Quills
• Fingernails
• Feathers

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Another Enigma
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How IS protein structure determined?

• What functions must protein structures serve?
• Structural Collagen, Keratin
• Catalytic Enzymes
• Receptors/Transporters Glucose, Insulin,
  Antibodies

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Three-Dimensional Structure

• Primary sequence of amino acids
• Secondary local spatial arrangement of the
  peptide backbone without regard to the
  conformations of the side chains
• Tertiary 3-D structure of an entire, single
  polypeptide
• Quaternary spatial arrangement of peptide
  subunits

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Three-Dimensional Structure

• The crystal diffraction patterns of pepsin
  provided the first evidence that proteins have a
  ordered array of atoms.
• Crystallography and 2D NMR are key in determining
  3-D structure.
• Bernal and Hodgkin, 1934

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Secondary Structure

• Helices
• Sheets
• Turns
• Depend on H-bonds between amide bond Hs and Os

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Peptide Bond Formation
O
R
NH3
C
CH
O-
NH3
CH
C
O-
O
R
H2O Condensation
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Tripeptide Structure
C-terminus
N-terminus
O
R
H
COO-
C
N
CH
NH3
CH
N
CH
C
O
H
R
R
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The Peptide Bond
O
R
H
COOH
C
N
CH
NH2
CH
N
CH
C
O
H
R
R
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Secondary Protein Structure
O
R
H
COO-
C
N
CH
NH3
..
..
CH
N
CH
C
O
H
R
R
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Canonical Peptide Bond Structures
-
O
O
C
C
or

N
N
H
H
Resonance or Tautomerism restricts peptide
bond rotation 40 double-bond character at C N
bond
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predominantly trans configurationabout the
peptide bond linkage
R
O
H
COOH
C
N
CH
NH2
CH
N
CH
C
O
H
R
R
Pro is the major exception 10 cis
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Primary Protein Structure
O
R
H
COOH
C
N
CH
NH2
CH
N
CH
C
O
H
R
R
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Secondary Structure(Spaghetti vs.
Ravioli)Lowest Energy Conformations Begin to
Direct Folding

• Steric Interactions
• Hydrophobic Interactions (Partitioning)
• Hydrogen Bonding (C?O---H-N or C?O---H-O)

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3-D Dependent on rotation about the C? dihedral
angles
R
H
O
?
COOH
C
N
C H
NH2
?
?
?
?
C H
N
C H
C
O
H
R
R
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? and ? are defined as 180 when peptide is fully
extended
R
H
O
?
COOH
C
N
C H
NH2
?
?
?
?
C H
N
C H
C
O
H
R
R
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Torsion Angles increase with clockwise
rotation when viewed from the ? - carbon
R
H
O
?
COOH
C
N
C H
NH2
?
?
?
?
C H
N
C H
C
O
H
R
R
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Steric interactions of carbonyl Os and amide
Ns of adjacent residues restrict rotations
R
H
O
?
COOH
C
N
C H
NH2
?
?
?
?
C H
N
C H
C
O
H
R
R
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Degrees of rotation for ? and ?

• Ramachandran Diagrams
• Map the possible rotations for each amino acid
• Some parts of the diagram (conformations) blocked
  by van der Waals distances between atoms

?
?
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Degrees of rotation for ? and ?
Approximate Degree of
Rotation C? - N C? - C ?-Helix 3.6 - 60
- 57 Sheet (antiparallel) - 140 135 Sheet
(parallel) - 119 113
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The Alpha Helix 1951, L. Pauling
Pitch 5.4 Å

Hydrogen binding between a. a. n and a. a. n4
Birkbeck College, UK http//www.cryst.bbk.ac.uk/P
PS95/course/3_geometry/helix2.html
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The Alpha Helix 1951, L. Pauling

• Right Handed
• 3.6 amino acids/turn 13 atoms 3.613 helix
• H-bonds between n and n4 amino acids every N-H
  and C-O are H-bonded
• Approximately 12 residues
• R-groups point outward
• Core has essentially no open space

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Helical Wheel Diagram
b
a
c
d
e
f
g
e
b
a
f
d
c
g
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Amino Acid Classification
NONPOLAR Ala (A) Val (V) Leu (L) Ile (I) Pro
(P) Phe (F) Trp (W) Met (M) Gly (G)
POLAR, UNCHARGED Ser (S) Thr (T) Cys (C) Gln
(Q) Tyr (Y) Asn (N) Gly (G)
CHARGE Lys (K) (10.54) Arg (R) (8.99) His (H)
(6.04)
R
- CHARGE Asp (D) (3.90) Glu (E) (4.07)
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Keratin Primary StructureA Pseudorepeating
Sequenceof 7 Amino Acids
KEY Yellow Hydrophobic/Nonpolar (e.g., V, L,
I, A) Green Hydrophilic/Charged (e.g., T, S, Q,
N)
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Helical Wheel Diagram(Tertiary and Quaternary
Structure)
g
e
e'
c
d
a'
c'
d'
a
b
f
b'
g
c
d
f
a
b
e
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?-KERATIN A Coiled Coil(7 amino acid
pseudo-repeat)
e
g
b
c
a
d
f
f
d
a
c
b
g
e
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Fibrous Proteins

• Keratin
• Silk Fibroin
• Collagen
• All have structural functions
• Protective
• Connective
• Supportive
• Single, dominant 2 structures

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?-KERATIN A Coiled Coil

• 30 different ?-keratins in mammals (?-keratins in
  birds reptiles) to form hair, nails, horn,
  epidermis
• 5.1 Å not 5.4 Å pitch helix 2-right-handed
  helices in left-handed coil with 7-residue
  pseudo-repeat
• Cysteine-rich Cys determines hardness, e.g.,
  nail versus callus skin
• Mercaptans (HSCH2CH2OH) can cleave Cys-Cys bonds
  and relax coils

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?-KERATIN A Coiled Coil

• Diamer two polypeptides in LH coil H to H
• Protofilament two rows of staggered diamers
• Protofibril two protofilaments
• Microfibril two protofilaments in parallel
• Macrofibril several microfibrils

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Secondary Protein Structure1951, L. Pauling
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The Beta Sheet 1951, L. Pauling

• 2 12 strands average is 6 strands
• 15 residues/strand
• Adopt pleated structure to optimize H-Bonding
• R-groups protrude from alternating sides of the
  sheet

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The Beta Sheet 1951, L. Pauling

• Right hand twist that is a compromise between
  H-bond and R-group interactions
• Parallel sheets require an out-of-plane loop
• Parallel are slightly less stable

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Beta-Sheet H-Bonding
Parallel Sheets
O C
O C
N - H
O C
N - H
N - H
O C
O C
N - H
O C
N - H
N - H
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Silk Fibroin A ?-Sheet

• Insects arachnidsextended antiparallel
  ?-sheets
• 6-residue repeat
• (-Gly-Ser-Gly-Ala-Gly-Ala-)n
• Strong, flexible, but not extensible due to
  extended sheets (peptide chain) structure

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Silk Fibroin A ?-Sheet
Ala
Gly
Ser
http//info.bio.cmu.edu/Courses/03231/ProtStruc/2s
lk.htm
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Collagen A Triple Helix

• Most abundant vertebrate protein
• 3-LEFT-handed helices in a RH superhelix with
  interchain H-bonding of Glys to Xs
• Gly-X-Y-Gly- repeat X is often Proline Y is
  often 3- or 4-Hydroxyproline and sometimes
  5-Hydroxylysine
• Unique a.a. composition 30 Gly 15-30
  Pro/HOPro

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Collagen A Triple Helix

• HO-amino acids formed as post-translational
  modifications
• Prolyl hydrolase requires Vitamin C (ascorbate)
• Opposite helix coiling lends to tensile strength
  under tension
• Inter-strand connections via Lys and His
• (Figure 16-9)

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Collagen Disease

• Scurvy Vit C deficiencyskin lesions,
  fragility, poor healing
• Lathyrism ingestion of sweet peareduced
  X-linking of HO-Lys due to inactivated lysyl
  oxidase -- bone, joints, large vessels
• Osteogenesis imperfecta reduced helix stability
  due to genetic Ala substitution of Gly
• Ehlers-Danlos syndromes lysyl oxidase and lysyl
  hydroxylase--India-Rubber man

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Collagen Crosslinking

• Collagen is synthesized as preprocollagen and
  exported from the cell for post-translational
  crosslinking
• Crosslinking sites are tissue dependent
• 2 Lys ? Allysine ? Allysine Aldol
• Allysine Aldol His ? Aldol-His HOLys ?
  4-amino acid complex
• ? Histidinodehydrohyroxymerodesmosine

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Nonrepetitive Structures

• Helix cappingAsn or Gln with a.a. 1-4
• Reverse turns or ?-turns (surface feature) 4
  a.a. forming a turn
• Omega (?) loops (surface feature) 6-16 a.a.s
  forming a surface loop
• No regular ? or ?
• ?-bulge extra a.a. in a ?- sheet
• Pro kinks in helices or sheets
• Large side-chains steric interactions

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Three-Dimensional Structure

• Secondary local spatial arrangement of the
  peptide backbone without regard to the
  conformations of the side chains
• Tertiary 3-D structure of an entire, single
  polypeptide, including the position of each atom
  in each amino acid side chain
• Quaternary spatial arrangement of peptide
  subunits

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Tertiary Structure

• X-ray Crystallography is a powerful tool
• Wavelength is approximately 1.5Å ? covalent bond
  distance. Wavelength determines resolution and,
  thus, produces a diffraction pattern determined
  by interaction with electron clouds in the
  molecule
• X-rays define electron space, excluding H atoms
  to provide a 3-D contour map

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X-ray Crystallography

• Protein crystals usually contain water of
  hydration
• Allows molecules to stay in a native-like
  structure in soft crystals
• Cause variation in the proteins diffraction
  pattern and makes individual atoms difficult to
  locate
• Usually does allows the shape of backbone to be
  clearly defined
• Knowledge of the proteins 1º sequences is needed
  to make sense of the diffraction pattern.

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Tertiary Structure Globular Proteins

• Lack long repeats like collagen and keratin
• Amino Acid polarities often defined their
  position in the molecule

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Globular Proteins

• Amino Acid Positions
• Val, Leu Ile, Met, Phe Hydrophobic interior
  (entropy driven exclusion of polar groups)
• Arg, His, Lys, Asp, Glu Hydrophilic exterior to
  accommodate charges
• Ser, Thr, Asn, Gln, Tyr Hydrophilic exterior or
  H-bonded in interior
• http//info.bio.cmu.edu/Courses/BiochemMols/ProtG/
  ProtGMain.htm

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Combining Secondary Structures

• Supersecondary Structural Motifs
• 1. ??? -- ?sheet-helix-sheet (parallel )
• 2. ? hairpin sheet-turn-sheet (antiparallel
  sheets)
• 3. ?? motif antiparallel helix-helix (keratin)
• 4. ? barrels multiple parallel or antiparallel
  sheets in barrel structures

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Structural Domains

• Two layers of secondary structure are usually
  required to sequester the hydrophobic core of a
  domain
• Domains usually interact to form binding pockets
  for substrates or cofactors
• Domains usually connected by hinges
• The overall structural domains define the
  conserved elements of evolution

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Hemoglobin
Quaternary Structures
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Quaternary Structures

• Hemoglobin
• ?1?2?1?2

?2
?1
?2
?1
http//jeffline.tju.edu/CWIS/DEPT/biochemistry/pro
tein/Hb/Hb1.JPEG
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Quaternary Structures

• Symmetries
• No inversion/mirror symmetry (would require
  D-a.a.s

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Protein Folding

• H-bonds
• Hydrophobic effects
• Ion pairs
• Disulfide Bonds
• Metal Ions coordinate bonds
• E.g., Zinc fingers
• Prosthetic Groups
• Heme

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Denaturation and Renaturation

• Conformationally-sensitive properties, e.g.,
  Optical rotation, viscosity, and UV absorption
  change abruptly with temperature and denaturation
• pH affects protein charge distribution
• Detergents are effective denaturants
• Oxidation/Reduction of environment
• Chaotropic Agents - urea guanidine HCl

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Renaturation

• Appropriate cysteine pairs may not match
• Some fold incorrectly because the order of
  folding does not match the folding order at the
  protein is synthesized on the ribosomes

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Native Folding

• 1. Secondary structures form
• 2. Hydrophobic Collapse yields molten globule
• 3. Subdomains formsmall local elements forming
  first
• 4. Hydrogen bonding and expulsion of water from
  the central core

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Folding ProteinsProtein Disulfide Isomerase
(PDI)

• Reduced PDI catalyzes rearrangement of
  established disulfides
• Oxidized PDI catalyzes the initial formation of
  disulfide bonds

SH
S
PDI
PDI
S
SH
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Folding ProteinsMolecular Chaperones

• Bind to proteins as they are synthesized to
  prevent premature folding
• Examples
• Hsp70
• Chaperonins (Hsp 60 Hsp 10) large, 7 subunit
  ATP driven

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Abnormal Folding

• ?-Amyloid Protein (Alzheimers Disease)
• Bovine Spongiform Encephalopathy, scrapie,
  Creutzfeldt-Jakob disease
• prion-based disease
• - prion causes misfolding.

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Protein Dynamics

• Flexibility and breathing may results in as
  much as 2 Å of displacement

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Quaternary Structure
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Family of O2-Binding Proteins

• Myoglobin heme group
• Leghemoglobin nitrogen-fixing bacteria in
  legumes
• Chlorocruorins annelids, altered porphyrin
  (greenish)
• Hemocyanin invertebrates, two Cu atoms no heme
• Hemerythrin invertebrates, Fe but no heme
• Hemoglobin heme group complex binding kinetics
  making it an Honorary Enzyme

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OO
H2CCH
CH3
H3C
CHCH2
Fe(II)
CH3
H3C
-OOC-CH2-CH2
CH-CH2-COO-
His F8
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Myoglobin Structure

• Intracellular in vertebrate muscle
• 153 residues in 8 helices (A-H)
• Single heme in hydrophobic pocket between helices
  E and F with Fe2 bond to 4 Ns of pyrrole, His
  F8 and an O2.
• Oxidation of Fe2 to Fe3 to form metmyoglobin or
  methemoglobin yields brown, oxidized product

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Myoglobin Function

• Oxygen STORAGE and TRANSPORT
• Relays O2 from capillaries to muscle cells
• Increases the effective solubility of O2
• Fully saturated over the pH range and conditions
  of the blood

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Myoglobin

• Small 153 a.a.s in 8 helices
• Single heme group porphyrin derivative
• One Fe(II) ligated by
• 4 pyrrole Ns
• One His F8 N
• One molecule of O2 held by His E7

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Myoglobin

• Fe (II) in the heme group gives the protein
  colors from dark purple to scarlet depending on
  oxygenation
• Oxidation of Fe(II) to Fe(III) yields
  metmyoglobin or methemoglogin, which are brown in
  color
• CO, NO and H2S bind the heme more tightly than O2
  and is the basis for their toxicity

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Myoglobin Function

• Storage/Oxygen Transport in Muscle
• Storage is more important in marine mammals where
  myoglobin is 10X higher
• Transport from capillaries to muscle cells,
  overcoming low solubility of O2, appears to be a
  predominant function in other mammals where
  diffusion is a limiting function for oxygenation
  of tissues

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Myoglobin Oxygen Transport

• Myoglobin Dissociation
• Mb O2 ? MbO2
• Kd Mb O2
• MbO2
• Thus, MbO2 is dependent upon O2 or pO2 which
  determines the
• YO2 fractional saturation of myoglobin

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Myoglobin Binding Curve
1.0
0.8
0.6
K p50 2.8 torr
YO2
0.4
0.2
0.0
5
10
15
20
25
30
pO2 (torr)
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Myoglobin Function
So if, p50 Myoglobin 2.8 torr And, pO2
arteries 100 torr pO2 veins 30 torr (760
torr 1 atm.) Then, Myoglobin 91 97
saturated, even at the capillaries ? efficient
transport to muscle
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Myoglobin Binding Curve
1.0
0.8
pO2 30 torr YO2 91
0.6
YO2
0.4
0.2
0.0
5
10
15
20
25
30
pO2 (torr)
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Hemoglobin Binding Curve
1.0
Myoglobin
0.8
Hemoglobin
For Hemoglobin, YO2 .55 _at_ 30 torr .95
_at_ 100 torr
0.6
YO Hb
0.4
0.2
0.0
20
40
60
80
100
120
pO2 (torr)
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Hemoglobin Binding Curve

• The sigmoidal binding curve is diagnostic for a
  cooperative binding system interaction of ? and
  ? subunits
• Changing slope means changing binding affinity
  for the first and last subunits

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Hemoglobin Binding Curve
1.0
Myoglobin
0.8
Hemoglobin
For Hemoglobin, YO2 .55 _at_ 30 torr .95
_at_ 100 torr
0.6
YO Hb
0.4
0.2
0.0
20
40
60
80
100
120
pO2 (torr)
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Hemoglobin Binding CooperativityExplaining the
T to R Transition

• Perutz determined a T (tense) and R (relaxed)
  state for OxyHb and DeoxyHb respectively using
  X-ray Crystallography
• O2 binding cause the Fe-Nporphyrin bonds to
  shorten, pulling Fe into a planar structure with
  the porphyrin ring
• Fe movement tilts Helix F down with translation
  across the heme plane

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OO
H2CCH
CH3
H3C
CHCH2
Fe(II)
CH3
H3C
-OOC-CH2-CH2
CH-CH2-COO-
His F8
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Hemoglobin Binding Cooperativity the T to R
Transition

• F-helix translation causes a 15 axial rotational
  shift between ?1-?2 and ?2-?1 subunits
• H-bonds and ion pairs between the ?1-?2 and ?2-?1
  subunits simultaneously snap to alternate (R)
  positions
• SUMMARY Binding of one O2 to one subunit causes
  a shift of all subunits from T to R increasing
  their affinity for O2

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The Bohr Effect

• Oxygen binding changes the pKs of amino acids in
  the hemoglobin ?N-termini and ?-C-termini due to
  altered ion pairing
• At physiological pH, Hb releases 0.6 protons
  per O2 bound
• Increasing the pH (removing protons) lowers the
  p50 of Hb (R) conversely, lowering the pH forces
  off oxygen (T)

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The Bohr Effect

• CO2 in the blood forms HCO3- H thus, lowering
  the p50 of Hb (carbonic anhydrase accelerates
  this Rx in erythrocytes)
• This allows additional oxygen delivery to the
  muscle when lactic acid builds up
• This also allows better oxygen release from Hb to
  Mb in the capillaries where CO2 is high

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Bisphosphoglycerate (BPG)

• Purified Hb has a greater oxygen affinity than Hb
  in whole blood
• Bancroft found that BPG binds only to DeoxyHb and
  helps to keep oxygen unbound once the Hb unloads
  oxygen and shifts to the T state in the
  capillaries
• BCG binds more tightly to adult than fetal HB to
  help deliver oxygen to the fetus

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Allosteric InteractionsSequential Model

• Ligand binding of one subunit affects neighboring
  subunit, but not all subunits uniformly
• If the mechanical coupling between subunits is
  very strong, conformation changes may occur
  simultaneously as in the symmetry model