Biochemistry Chapter 7 Protein Function and Evolution

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BiochemistryChapter 7Protein Function and
Evolution

• Mak Oi-Tong

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Introduction (Figure 7.1)

• To look more closely at how protein structures
  are related to the molecules functions and how
  they may have evolved to fulfill the protein
  functions.
• Globin myoglobin (Mb) and hemoglobin (Hb)
• They play important roles in animal metabolism,
  particular for the utilization of oxygen.

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The role of hemoglobin and myoglobin

• Myoglobin is used to store oxygen from Hb in
  muscle, and hemoglobin is used for transportation
  of oxygen to tissues.
• Both Mb and Hb have a common structural motif.
• Mb is monomeric and Hb is tetrameric protein.
• Both Mb and Hb have a prosthetic group, the heme,
  which contains the oxygen binding site.

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Fig. 7.1
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Fig. 7.3
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The mechanism of oxygen binding by heme proteins

• The oxygen binding site
• In Mb and Hb, the heme contains the ferrous iron
  (Fe2) coordinated in a protoporphyrin IX which
  is a main class of porphyrin.
• The ferrous ion is coordinated with the four
  nitrogen atoms of porphyrin, one with histidine
  residue No. 93 and one with O2. When binding with
  O2 , (oxymyoglobin) the O2 lies another histidine
  64.
• Instead of O2, the heme can bind CO, similar
  size of , with much greater affinity than the O2,
  and the binding is irreversible, and CO is a
  toxic gas.

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Fig. 7.4
FiO2 O2
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Analysis of oxygen binding by myoglobin

• Gas partial pressure (PO2) is the expression of
  oxygen concentration of any gas dissolved in a
  fluid.
• The oxygen binding curve for myoglobin (Fig. 7.6)
  has a hyperbolic shape.
• See pp 217 for fraction of the Mb sites that have
  oxygen bound to them (?) depend on the
  concentration (partial pressure) of free oxygen.
• The equilibrium constant K is called association
  constant or affinity constant.
• T PO2 / (P50 PO2) (Equ. 7.6)
• K k1 / K-1
• k1 the binding reaction and K-1 the release
  reaction constant.

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Fig. 7.6
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Fig. 7.7 Dynamics of oxygen Release by myoglobin
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Oxygen transport Hemoglobin

• Hb is called oxygen transport protein.
• Hb accepts O2 from lung capillaries and delivers
  to the oxygen binding protein Mb.
• The binding affinity of oxygen to Hb is quite
  different from Mb.
• Structural differences Mb a monomer and Hb a
  tetramer.

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Cooperative binding and allostery

• A situation in which the binding of one
  constituent to a macromolecule favors the binding
  of another, e.g. hemoglobin cooperatively binds
  to oxygen molecules.
• The macromolecules are normally multisubunit
  structure.
• The switch from a weak binding state to a strong
  binding state can be shown by the Hill plot.

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Fig. 7.8
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• T / (1- ?) PO2 / P50 (rearranged of equ. 7.6)
• log ? / (1- ?) log PO2 log P50
• y mx c , m slope 1
• log ? / (1- ?) 0 (? 0.5, ie. 50
  saturation)
• log PO2 log P50 PO2 P50
• For Hb, the cooperative binding from weak binding
  state (high P50) to strong binding state (small
  P50), and produce a different slope between the
  parallel lines.
• The different slope is called Hill coefficient, a
  number for the binding affinity.
• The cooperative effect is called allosteric
  effects, the binding of one ligand influences the
  binding of another ligand, and the ligands may be
  the same as the substrate or different from the
  substrate.

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Fig. 7.9
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Model for the allosteric change in hemoglobin

• Sequential models by Koshland, Nemethy and
  Filmer.
• The subunits can change their conformation one at
  a time, and the presence of some subunits
  carrying O2 favours the strong-binding oxy form,
  containing tense (T) state and relaxed (R) forms.
• Concerted models by Monod, Wyman and Changeux.
• The entire hemoglobin tetramer exists in an
  equilibrium between two forms, Tense (T) and
  relaxed (R) forms.
• The shift between two forms is a concerted one.

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Fig. 7.10
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Change in hemoglobin structure accompanying
oxygen binding

• Hb of higher vertebrates is made up of two types
  of chains, a- and ß-chain, and their sequences
  are similar.
• Essential residues like the proximal and distal
  histidine, F8 and E7, are conserved and their
  tertiary structures are conserved too.
• When Hb is in concentrated urea solution, it
  dissocites into aß dimers, suggesting aß contains
  the closest and strongest contacts.
• Change of conformation of hemoglobin in
  oxygenation, refers to Fig. 7.12.

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Fig. 7.11
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Fig. 7.12a
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Fig. 7.12b
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A closer look at the allosteric change in
hemoglobin

• By X-ray diffraction, the overall change of deoxy
  and the oxy state of Hb can be formulated.
• Mechanism of the T to R transition in hemoglobin
  is shown in Fig. 7.13.
• By using site-directed mutagensis method, the
  proximal histidine is replaced by a glycine,
  substitute with imidazole, it does not move the F
  helix. (Fig. 7.14)

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Fig. 7.13
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Fig. 7.14
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Effects of other ligands on the allosteric
behavior of hemoglobin
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Response of pH changes The Bohr effect

• Lower pH in blood capillaries has the effect of
  lower the oxygen affinity of hemoglobin for more
  release of the last trace of oxygen The Bohr
  effect.
• When proton concentration increases, it will
  promote the release of oxygen by driving the
  reaction to the right as the following
• Hb4O2 nH lt gt Hb.nH 4O2
• A decrease of only 0.8 unit shifts the P50 from
  about 20 to over 40 mm Hg, nearly double the
  oxygen unload to myoglobin.

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Fig. 7.16
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Carbon dioxide transport

• Some of the carbon dioxide becomes bicarbonate,
  releasing protons that contribute to Bohr effect.
• CO2 itself reacts directly with hemoglobin,
  binding to the N-terminal amino groups of the
  chains to form carbamates.
• The negative charged group is formed at the
  carbamate, stabilizing the salt bridge formed a
  and ß chains which is favoured deoxy form (T).
• Hyperventilation, a lack of CO2 stimulation of O2
  released.

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Effect of bisphosphoglycerate

• 2,3-Bisphosphoglycerate (BPG) can, lower the
  oxygen affinity of Hb as CO2 done.
• Activation of BPG in Hb is that it will narrow
  the cleft of oxy form when it binds to Hb, and
  decreases on O2 affinity to oxy-Hb.
• Similar effect in heavy smokers.
• BPG also have different binding effect in mother
  Hb (HbA) and fetus Hb in which containing
  a2?2,HbF.
• Results give lower O2 affinity in HbA then HbF,
  and oxygen can transport from mother blood to
  fetus blood.
• Other mammals use inositol hexaphosphate (bird)
  or ATP (fish) for the similar effects of BPG.

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Fig. 7.17
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Fig. 7.18
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Fig. 7.19
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Protein evolution Myoglobin and Hemoglobin as
examples
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Structure of eukaryotic genes exon and introns

• Eukaryotic genes are discontinuous, containing
  both expressed regions (exons) and regions not
  expressed as protein sequence (introns).
• Pre-mRNA is present.

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Fig. 7.20
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Mechanism of protein mutation

• Replacement of on DNA base by another
• Missense mutation
• Nonsense mutation, the codon for an amino acid is
  replaced by a stop codon.
• Nucleotide deletions or insertions
• Frameshift mutation results in a complete change
  in the amino acid sequence in the C-terminal
  direction from the point of mutation.
• Gene duplication and rearrangement
• Gene duplication
• Gene fusion
• Gene recombination

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Fig. 7.21
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Evolution of the myoglobin-hemoglobin family of
proteins

• 25 amino acids change in 100 millions years, and
  4 millions years for each AA.
• If the evolution is correct, both Mb and Hb come
  from the same ancestor.
• The family tree of Hb and Mb and the functions of
  different gene products (Fig. 7.22 and 7.23).
• Two important events happened at 800 and 500
  millions years ago for the gene was duplicated
  into Mb and Hb, and then to a and ß chains.
• Although most of the AA residues have changed,
  the secondary and tertiary structure remain
  unchanged, the basic globin fold (Fig. 7.24).

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Fig. 5.14
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Fig. 7.22
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Fig. 7.23
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Fig. 7.24
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Hemoglobin variants Evolution in
progress
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Variants and their inheritance

• Some abnormal hemoglobin are present in human
  because of the mutation by continuing evolution.
  Some are neutral or harmful, and some are fatal
  and pathologies (Fig.7.25).
• An individual can have 3 possible combinations of
  Hb genes
• ß ß homozygous---normal type
• ß ß Heterozygous---mixed type
• ß ß homozygous---the variant type
• Inheritance of normal and variant protein in a
  heterozygous is shown in Fig. 7.26.

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Fig. 7.25
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Fig. 7.26
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Pathological effects of variant hemoglobin

• Mutation in human hemoglobin, see Table 7.2.
• Results
• change of O2 affinity
• loss of heme
• dissociation of tetramer and
• change of RBC shape, sickling.

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Sickle-cell anemia

• Anemia means less effective in transporting O2.
• Sickle-cell hemoglobin (anemia)
• Exist in deoxygenated state because of the
  rodlike structure.
• Block capillaries, and causing inflammation and
  pain.
• More seriously, the sickle Hb will be fragile and
  cause death.
• Molecularly, sickle-cell Hb is caused by the
  change of glutamic acid (Glu6) in ß chain with a
  valine, and causes the change of Hb conformation.
• High incidence area of sickle-cell disease is
  related to the high incidence of malaria, because
  of higher resistance to malaria.

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Thalassemia

• One or more of the Hb genes are not producing or
  missing.
• All genes may be present, because of a nonsense
  mutation that produces a nonfunctional chain.
• All genes may be present, but a mutation has
  occurred outside the coding region that the
  protein chain is not produced or functional well.
• ß -Thalassemia has no ß chain produced, only
  fetal ? chain produced. ß is that the ß gene is
  partially inhibited.
• a -Thalassemia has no a chain produced,
• Compared the 2 copies ofa genes and 1 copy of ß
  gene present, advantages of gene duplication with
  two copies of a gene than on copy of ß gene.

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Immunoglobulins

• Variability in structure yields versatility in
  binding

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The immune response

• Classification
• Humoral immune response by B lymphocytes to
  produce antibodies.
• Cellular immune response by T lymphocytes.
• Antigen and antibody
• Antigenic determinant (epitope)
• AIDS (acquired immune deficient syndrome) is a
  kind of disease that destroys the defense of
  human body, the breakdown of immune system.
• Characteristics of immune response versatile or
  variable and having memory for further reaction.

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Fig. 7.29
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Clonal selection theory

• Instructive theories suggested that an antigenic
  determinant could induce an antibody molecule to
  a particular tertiary folding, and the suggestion
  is wrong.
• B stem cells in bone marrow differentiate to
  become mature lymphocytes, and each producing a
  single type of antibody, each type with a binding
  site that will recognize a specific molecular
  shape (antigen).
• Binding of an antigen to one of these antibodies
  stimulates the cell carrying to replicate,
  generating a clone aided by helper T cells.
• Two classes of cloned B cells are produced,
  effector B cells produce soluble antibodies which
  are secreted into blood circulatory system, and
  memory cells that persist for some time. It
  allows a rapid secondary response.
• Autoimmunity is the production of antibodies
  against our normal tissues.

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Fig. 7.30
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Fig. 7.31
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Structure of antibodies

• Five different classes of antibodies, Ig (MAGED).
• Functions and properties of antibodies see Table
  7.3.
• Structure of antibody
• Containing 2 heavy chains (53000) and 2 light
  chains (23000) of which held by disulfide bonds.
• Constant domains which are identical in all
  antibodies of a given class.
• Variable domains in the difference of amino acid
  sequence, and having 2 binding sites for a normal
  antibody.
• The hinge regions can be cleaved to produce Fab
  fragments, containing only one binding site.
• Binding sites are at the extreme end of the
  variable domains.
• The constant domains of the hinge chains in the
  Y-shaped molecule is function as effecters to
  signal macrophage to attack particles or cells
  labeled by antibody binding, the basic function
  of B cell antibodies.

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Fig. 7.32
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Fig. 7.33
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Generation of antibody diversity

• The human does not have enough room to code
  (genes to products) for each of different Ig
  molecules.
• By two special processes
• Recombination of exons the genome contains
  libraries of exons corresponding to different
  portions of Ig molecules, and can rearrange to
  create different combinations of Igs, over 100
  millions Igs can be produced.
• By somatic (body) mutation caused by unusual high
  rate of mutation in the certain portions of
  variable regions of Ig molecules.

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T cells and the cellular reponse

• Cellular response involves in tissue rejection
  and destroying virus-infected cells.
• T cells are also called killer T cells and they
  contain similar Fab fragments of antibody
  molecule as surface receptors, can bind of short
  oligopeptides.
• The binding of T cells receptor must be helped by
  another class of Ig like molecule called MHC
  (major histocompatibility complex).
• When T cells bind to the foreign cells, they will
  release a protein called perforin to kill foreign
  cells.
• The basic binding of T cells to the target cells
  is based on the Ig molecule fold

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Fig. 7.35
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Fig. 7.36
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Aids and the immune response

• AIDS (acquired immune deficiency syndrome) is a
  disease of the immune system.
• It is caused by the human immunodeficiency virus
  or HIV which attacks a class of helper T cells.

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Fig. 7.37