Protein Binding Phenomena

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Protein Binding Phenomena

• Lecture 7, Medical Biochemstry

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Ligands

• Ligand - any molecule that can bebound to a
  macromolecule (e.g., a protein). Examples of
  ligands include molecules ranging from small
  organic metabolites like glucose or ATP to large
  molecular polymers like glycogen or proteins. For
  our purposes, any molecule that can bind to a
  protein can be termed a ligand

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Why do ligands bind proteins?

• Both ligand and protein are surrounded by a water
  solvent shell. Each is undergoing random thermal
  motions that can lead to randomly oriented
  collisions between the protein and the ligand.
  Except for v. large ligands, thermally driven
  diffusion of the ligand is much more rapid than
  that of the protein.

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Ligand Binding (cont)

• Both protein and ligand can have functional
  groups such as hydroxyl, carboxyl, amino, amide
  and alkyl groups in various degrees of contact
  with the aqueous solvent. These functional groups
  on the ligand and on the protein may be capable
  of forming non-covalent bonds with each other.

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Ligand Binding (cont)

• If these functional groups are oriented during a
  collision so that they are spatially near one
  another, binding may occur. Of course, different
  ligands will have different spatial orientations
  of these functional groups and therefore will
  require different configurations of functional
  groups on the surface of the protein to permit
  binding. This matching of functional group
  spatial orientations are what determines the
  specificity of binding to that particular
  protein. Each protein will have its own
  characteristic set of binding specificities.

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Lock and Key Binding Model
For this model, the shapes of the surfaces of
both protein and ligand must fit like a lock and
key otherwise steric hindrance will prevent
binding
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Induced Fit Binding Model
For this model, a loosely bound ligand can
interact with functional groups on the protein
and cause the protein to alter its conformation
so as to better fit and bind the ligand more
tightly (hence induced fit). This can be thought
of as a stabilization of a particular protein
conformation by ligand binding.
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Hexokinase - Induced Fit Example
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Compare
Bond kJ/m
C-C 350
C-H 410
O-H 460
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Examples of H-bonding
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Ligand Binding - Ionic Interactions (Ex
ATP-Mg-Arg)
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Scatchard Equation
A mathematical model of binding phenomena
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Scatchard Equation and Graph Determination of Kd
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The Significance of Kd

• The tighter the binding of a ligand to a protein,
  the smaller the Kd value (e.g. pM values) and the
  less likely that the ligand will dissociate from
  the protein once they are bound together. For a
  weak Kd value, the concentration is much higher
  (e.g., mM). These statements are made assuming
  that tighter binding is the desired property. Kd
  values are frequently used for comparisons of
  binding between different classes of ligands to a
  protein (as in comparing different drugs).
  Similarly, the Kd for one ligand can be compared
  for binding to many different receptors on the
  same cell or different cell types or species.

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EXAMPLE
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Ligand Binding ExampleIntracellular Signalling
Cascades

• The Kd for binding of ligands (like growth
  factors or hormones) to their specific receptors
  is generally very tight with nanomolar or
  picomolar values. As the signalling cascade
  proceeds, the Kd values progressively increase
  (weaker binding) to micromolar values after
  production of cAMP. It is the very low Kd values
  of the receptor-ligand interactions that dictate
  the specificity of any signalling cascade. This
  is important for cellular function in that the
  cascades only become activated in response to
  specific ligand-receptor interactions.

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Myoglobin Hemoglobin
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Heme Structure
Protein-Heme Complex with bound oxygen
Heme-Fe2
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Myoglobin Properties

• At the tertiary level, surface residues prevent
  one myoglobin from binding complementarily with
  another myoglobin thus it only exists as a
  monomer.
• Each monomer contains a heme prosthetic group a
  protoporphryin IX derivative with a bound Fe2
  atom.
• Can only bind one oxygen (O2) per monomer
• The normal physiological O2 at the muscle is
  high enough to saturate O2 binding of myoglobin.

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Hemoglobin Properties

• At the tertiary level, the surface residues of
  the a and b subunits form complementary sites
  that promote tetramer formation (a2b2), the
  normal physiological form of hemoglobin.
• Contains 4 heme groups, so up to 4 O2 can be
  bound
• Its physiological role is as a carrier/transporter
  of oxygen from the lungs to the rest of the
  body, therefore its oxygen binding affinity is
  much lower than that of myoglobin.
• If the Fe2 becomes oxidized to Fe3 by chemicals
  or oxidants, oxygen can no longer bind, called
  Methemoglobin

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Oxygen Saturation Curves

• Useful analyses of myoglobin and hemoglobin
  functions have resulted from plotting the
  fraction of protein with bound O2 (fractional
  saturation) versus the concentration of O2
  (partial pressure, p)
• For myoglobin, a hyperbolic line results that
  reflects the high affinity of myoglobin for O2
  binding
• For hemoglobin, the curve is sigmoidal (S-shaped)
  and reflects the average affinity of the four
  subunits for O2 binding.

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Oxygen Saturation Curves for Myoglobin
Hemoglobin
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Hill Equation and Cooperativity
An empirical fractional saturation equation from
the oxygen curves can be derived based on the
data as follows
Taking the log of this equation and rearranging
results in the following Hill Equation
The slopes of the resulting straight line curves
are an indication of cooperativity in binding of
oxygen
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The Hill Plot
Cooperativity Index n 1, no cooperativity in
binding, as seen for myoglobin n gt 1, positive
cooperativity binding of ligand to one subunit
increases the affinity of a second site for
binding, and so on, as in hemoglobin
n lt 1, negative cooperativity binding of ligand
to one site decreases the affinity for
binding to a second site
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Hemoglobin Sub-unit Types

• Alpha-like (a)
• 1. a major adult form
• 2. z (zeta) embyronic form
• Beta-like (b)
• 1. b major adult form
• 2. d minor adult form
• 3. g major fetal form
• 4. e embyronic form

Note Complex genetic control mechanisms
discussed later in the course are responsible
for turning on and turning off the expression of
hemoglobin during development
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Developmental Expression of Hemoglobin Sub-units