Proteins: myoglobin Mb

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Proteinsmyoglobin (Mb) hemoglobin (Hb)

• Belinda Sharpe
• b.k.sharpe_at_sussex.ac.uk

2
Books

• Standard Biochemistry Books (Voet Voet or
  Stryer or Horton)
• Introduction to protein structure Branden
  Tooze
• Structure and mechanism in protein science
  Fersht
• Foundations of chemical biology Dobson, Gerrard
  Pratt
• I wish Id made you angry earlier Perutz

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What does Hb do?

• Ultimately, Hb is an O2-transport protein.
  However, this statement requires a subtle
  qualification, because Hb is an exquisite
  O2-delivery system. More of this later. During
  the course of evolution Hb has adapted to
  performing its O2-delivery role in fish, mammals
  and birds in varied environments. Along the way,
  Hb has been recruited to other tasks
• Hb is a temperature controller. O2 binding to Hb
  is (usually) exothermic that is, heat is given
  out. This means that when oxyHb arrives at
  muscle, heat is required to liberate O2. Whilst
  this isnt generally a problem to humans, it is
  for animals from colder environments where heat
  is more precious. Hbs in these animals have
  evolved to reduce the heat needed to free oxygen,
  and in some cases (tuna) heat is even evolved in
  the process. At the other extreme, in the
  heavily worked flight muscles of some birds,
  efficient heat loss is essential to avoid
  overheating. Here O2 release requires 3 times as
  much heat as it does in man. Finally,
  differences in the thermodynamics of O2 binding
  to HbA and HbF possibly provide a mechanism for
  cooling the well-insulated foetus in pregnancy.
• Hb is a provider of ballast. Some fish use
  gaseous O2 for buoyancy a pH change triggers O2
  release from oxyHb into the swim bladder.

4
Why bother with all that protein stuff?

• O2 solubility. The solubility of O2 in blood is
  low, 0.1 mM, and diffusion would not cover the
  demand for O2 in the body. O2 concentrations in
  blood plasma are increased 100 fold by O2
  binding to Hb.
• O2 oxidises Fe2 to Fe3. Four iron atoms lie at
  the heart of the Hb structure. Maintenance of
  these in the Fe(II) oxidation state (2) is
  critical for O2 binding Fe(III) Hb (metHb) does
  not bind O2. The protein scaffold protects Fe
  against oxidation.
• CO and NO poison Hb. Molecules similar to O2 can
  act as poisons by binding to the Fe atoms of Hb.
  The protein structure of Hb hinders binding of
  alternative molecules and, so, specifies the
  preference for O2.
• Control is needed. The 3D-structure of Hb, which
  is determined by its amino acid sequence, is able
  to adjust in response to subtle changes in its
  environment releasing or binding O2 where
  appropriate. Moreover, small changes to the
  proteins primary structure allow exquisite
  tailoring protein function.
• e.g. HbA versus HbF
• e.g. loss of control in sickle cell Hb (HbS).

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The structure of Hbintroducing the players
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1. The polypeptide chains

• The ?-chain of human Hb has a unique amino acid
  sequence, or primary structure of 141 residues
• VAL LEU SER PRO ALA ASP LYS THR ASN VAL LYS ALA
  ALA TRP GLY LYS VAL GLY ALA HIS ALA GLY GLU TYR
  GLY ALA GLU ALA LEU GLU ARG MET PHE LEU SER PHE
  PRO THR THR LYS THR TYR PHE PRO HIS PHE ASP LEU
  SER HIS GLY SER ALA GLN VAL LYS GLY HIS GLY LYS
  LYS VAL ALA ASP ALA LEU THR ASN ALA VAL ALA HIS
  VAL ASP ASP MET PRO ASN ALA LEU SER ALA LEU SER
  ASP LEU HIS ALA HIS LYS LEU ARG VAL ASP PRO VAL
  ASN PHE LYS LEU LEU SER HIS CYS LEU LEU VAL THR
  LEU ALA ALA HIS LEU PRO ALA GLU PHE THR PRO ALA
  VAL HIS ALA SER LEU ASP LYS PHE LEU ALA SER VAL
  SER THR VAL LEU THR SER LYS TYR ARG
• This sequence determines the 3-D, or tertiary
  structure of the ?- chain, which in combination
  with the ?-chain determines the function of Hb.
• In other words Protein sequence dictates
  structure, dictates function.

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2. The globin fold
This tertiary structure comprises 8 a-helices,
which are examples of secondary
structure HELIX-1 SER-3 to GLY-18 HELIX-2
HIS-20 to SER-35 HELIX-3 PHE-36 to
TYR-42 HELIX-4 HIS-50 to GLY-51 HELIX-5 SER-52
to ALA-71 HELIX-6 LEU-80 to ALA-88 HELIX-7
ASP-94 to HIS-112 HELIX-8 THR-118 to SER-138
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3. The heme group
This is an example of a protein prosthetic group.
It is a porphyrin ring with an Fe atom at its
centre.
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The heme in more detail
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4. The quaternary structure of Hb
Hb is a tetramer an ???? heterotetramer
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Structural principleshow do proteins fold up?
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Principles for protein folding Anfinsens
experiment
In other words the primary sequence of a protein
dictates its 3-D structure and, as a result, its
function.
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How does this sequence dictate the a-chain
tertiary structure?
VAL LEU SER PRO ALA ASP LYS THR ASN VAL LYS ALA
ALA TRP GLY LYS VAL GLY ALA HIS ALA GLY GLU TYR
GLY ALA GLU ALA LEU GLU ARG MET PHE LEU SER PHE
PRO THR THR LYS THR TYR PHE PRO HIS PHE ASP LEU
SER HIS GLY SER ALA GLN VAL LYS GLY HIS GLY LYS
LYS VAL ALA ASP ALA LEU THR ASN ALA VAL ALA HIS
VAL ASP ASP MET PRO ASN ALA LEU SER ALA LEU SER
ASP LEU HIS ALA HIS LYS LEU ARG VAL ASP PRO VAL
ASN PHE LYS LEU LEU SER HIS CYS LEU LEU VAL THR
LEU ALA ALA HIS LEU PRO ALA GLU PHE THR PRO ALA
VAL HIS ALA SER LEU ASP LYS PHE LEU ALA SER VAL
SER THR VAL LEU THR SER LYS TYR ARG
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The 20 amino-acid residues
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Lecture 2
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How does this sequence dictate the a-chain
tertiary structure?
VAL LEU SER PRO ALA ASP LYS THR ASN VAL LYS ALA
ALA TRP GLY LYS VAL GLY ALA HIS ALA GLY GLU TYR
GLY ALA GLU ALA LEU GLU ARG MET PHE LEU SER PHE
PRO THR THR LYS THR TYR PHE PRO HIS PHE ASP LEU
SER HIS GLY SER ALA GLN VAL LYS GLY HIS GLY LYS
LYS VAL ALA ASP ALA LEU THR ASN ALA VAL ALA HIS
VAL ASP ASP MET PRO ASN ALA LEU SER ALA LEU SER
ASP LEU HIS ALA HIS LYS LEU ARG VAL ASP PRO VAL
ASN PHE LYS LEU LEU SER HIS CYS LEU LEU VAL THR
LEU ALA ALA HIS LEU PRO ALA GLU PHE THR PRO ALA
VAL HIS ALA SER LEU ASP LYS PHE LEU ALA SER VAL
SER THR VAL LEU THR SER LYS TYR ARG
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The globin fold
This tertiary structure comprises 8 a-helices,
which are examples of secondary
structure HELIX-1 SER-3 to GLY-18 HELIX-2
HIS-20 to SER-35 HELIX-3 PHE-36 to
TYR-42 HELIX-4 HIS-50 to GLY-51 HELIX-5 SER-52
to ALA-71 HELIX-6 LEU-80 to ALA-88 HELIX-7
ASP-94 to HIS-112 HELIX-8 THR-118 to SER-138
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The 20 amino-acid residues
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Amphipathic a-helices
VAL LEU SER PRO ALA ASP LYS THR ASN VAL LYS ALA
ALA TRP GLY LYS VAL GLY ALA HIS ALA GLY GLU TYR
GLY ALA GLU ALA LEU GLU ARG MET PHE LEU SER PHE
PRO THR THR LYS THR TYR PHE PRO HIS PHE ASP LEU
SER HIS GLY SER ALA GLN VAL LYS GLY HIS GLY LYS
LYS VAL ALA ASP ALA LEU THR ASN ALA VAL ALA HIS
VAL ASP ASP MET PRO ASN ALA LEU SER ALA LEU SER
ASP LEU HIS ALA HIS LYS LEU ARG VAL ASP PRO VAL
ASN PHE LYS LEU LEU SER HIS CYS LEU LEU VAL THR
LEU ALA ALA HIS LEU PRO ALA GLU PHE THR PRO ALA
VAL HIS ALA SER LEU ASP LYS PHE LEU ALA SER VAL
SER THR VAL LEU THR SER LYS TYR ARG
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What stabilises folded structures?
1. Hydrogen bonds
2. The hydrophobic effect
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A walk througha globin
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How do proteins fold in mechanical terms? The
Levinthal paradox.

• How long would it take to fold a protein via a
  random walk through conformational space?
• These could all be in a-helical conformations, b-
  and any combination of a- and b- in between.
• I.e. there are TWO conformations per amino acid

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And for a real protein..

• Consider RNAse A with 123 amino acids
• 2123 1 x 1037 conformations
• 1013 conformations can be examined per second
• Therefore, time to test all conformations of
  RNAse A (1 x 1037)/(1 x 1013) 1 x 1024
  seconds. BUT
• The Universe is only estimated to be 1 x 1017 s
  old.

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Lecture 3
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Back to the chase.
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Oxygen-binding to Hb

• Summary
• Sigmoidal
• Cooperative
• Low affinity in the veins
• High affinity in the arteries
• p50 25 mm/Hg

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The structures of Mb Hb compared
Myoglobin (Mb)
Hemoglobin (Hb)
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O2-binding to Mb Hb compared

• Summary of O2-binding to Mb
• Hyperbolic
• Non-cooperative
• High affinity for O2 andmuch higher than that of
  Hb
• p50 1 mm/Hg

Hb
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The structures of Mb Hb compared

• Mb
• One polypeptide chain
• One heme, one Fe, one 02
• Tertiary structure only (like b of Hb)
• No cooperativity

• Hb
• Four polypeptide chains
• Four heme, four Fe, four 02
• Tertiary and quaternary structure
• Cooperativity

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In biological terms, why is Mbs affinity for O2
higher than that of Hb?
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What is cooperativity?
Adair fitted this curve mathematically assuming
four independent binding constants. The
resulting affinity of Hb for the 4TH oxygen was
1000 greater than that for the first. This is
cooperativity. Monod, Wyman Changeux proposed
a model (the MWC or concerted model) that
provided a structural rationale for this
behaviour and led to an understanding of control
in O2 binding by Hb. See AR Fersht, for an
alternative (the KNF model).
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The MWC Model

• Assumptions of the MWC model
• the protein is an oligomer
• the protein has two structural forms (tense, T,
  and relaxed, R)
• the T state has a lower affinity for O2
• all binding sites in each state (T or R) are
  equivalent.

This idea that multidomain/multimeric proteins
can exist in two different states is known
as ALLOSTERY which means OTHER SHAPE
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The evidence for allostery

• monomers of Hb bind O2 like Mb
• ?? homotetramers show no cooperativity
• X-ray crystallography

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The mechanism of allostery in Hb 1. Triggering
the structural change
distal His
proximal His
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The Fe movesslightly
oxy Hb
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The mechanism of allostery in Hb 2. Propagating
the structural change
The F-helix moves a littles
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Schematic Diagrams
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The quaternary structure changes

• Upon oxygen binding
• a-b subunit salt bridges break
• a1b1 twists relative to a2b2
• heme-heme distance reduces
• central cavity constricts
• In short, the Tense deoxy state relaxes and
  switches to the Relaxed oxy state. These
  changes transmits the structural changes to the
  other heme groups and INCREASES their O2 binding.
  This is cooperativity.

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Hb - the movie
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Cooperativity restated
The structural changes that lead to subunit
movements, the breaking of salt bridges and so on
ALL COST ENERGY. But this energy is almost all
paid upon binding the first O2. This weakens the
binding of the first O2. But subsequent binding
events, which do not to pay the toll, are eased
and strengthened as a consequence.
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Control 1. BPG, a chemical effector
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(No Transcript)
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Control 2. The Bohr effect

• Metabolically active tissues are rich on CO2 and
  H.
• At such sites Hb releases O2 and picks up CO2 and
  H.
• Why?

These additional charges form additional salt
bridges to further cross-link the Hb quaternary
structure and stabilise the T state. Hence, they
lead to the release of O2.
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Control 3. Sequence changes,HbF versus HbA

• HbF is a2g2 c/f a2b2 of HbA
• One of the changes in the g chain vs the b chain
  is His143Ser, which lies in the central cavity.
• This changes lowers the affinity of deoxyHbF for
  BPG relative to deoxyHbA
• This increases the affinity of HbF for O2.

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Control 4. Selective binding,O2 versus CO and NO
Schematic diagrams
distal His
proximal His
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Loss of control More sequence changes,HbS
versus HbA