Protein Function

1
Protein Function

• Want to explore how a proteins structure relates
  to how it interacts with other molecules.
• The function of many proteins involves the
  reversible binding of some ligand to the protein.
  In many cases the ligand is a small molecule
  however, it can also be another protein.
• The binding of a ligand to a protein involves
  non-covalent forces, which enables the
  interactions to be transient.
• The site on the protein where the ligand binds is
  complementary to the ligand in size, shape,
  charge, or hydrophobic / hydrophilic character.
  This site is called the binding site.
• Typically, a protein is very specific for any
  particular ligand that it characteristically
  binds, being able to discriminate among ligands
  that are similar in shape, size, or charge.
• Proteins are not rigid entities, but are flexible
  in nature. They breathe. They can undergo
  subtle conformational changes or very large
  conformational changes to adapt to the structure
  of the ligand, allowing tighter binding. This
  adaptation is called induced fit.
• Reading Lehninger Chap 7, pp. 203-221.

2
Myoglobin Hemoglobin

• Myoglobin and hemoglobin are two of the
  most-studied and best-understood proteins in all
  of biochemistry. These are the first two
  proteins to have their three-dimensional
  structures solved by x-ray diffraction methods,
  which represented a milestone in biochemistry.
• These proteins were crucial in the conversion of
  anaerobic life to aerobic life. Aerobic
  metabolism yields much more energy than anaerobic
  metabolism. Allowed for more complex life forms
  to evolve. Oxygen evolving photosynthetic
  organisms and plants were crucial in this
  evolutionary process.
• Problem. Oxygen (Dioxygen) has limited
  solubility in water. It is in fact a hydrophobic
  molecule. Need a way to bind oxygen effectively,
  in order to deliver it to tissues in sufficient
  quantities.
• Proteins like myoglobin and hemoglobin evolved
  to fulfill that purpose.

3
Heme Prosthetic Group

• Oxygen does not bind well to any of the amino
  acid side chains of the standard 20 amino acids.
  
• Oxygen does bind to various metals, including
  iron and copper.
• In the cell, iron is frequently found in a
  sequestered state as part of a complex with
  protoporhyrin IX.
• A porphyrin is an organic macrocycle that is
  composed of four pyrrole rings that are linked by
  methene bridges. They differ in the type and
  degree of substitution about the macrocycle.
  Protoporphyrin IX is only one example of a
  porphyrin.
• Note that the free porphyrin is flat, and that
  there are two open sites for binding to the iron.
  
• A porphyrin is considered to be a prosthetic
  group or cofactor, which is a small molecule that
  the protein employs to aid it in carrying out its
  function. There are many different types of
  prosthetic groups or cofactors.

4
Oxygen Binding to Heme

• Protoporphyrin IX containing a bound iron atom is
  called a heme. The iron within the heme
  prosthetic group is referred to as heme iron.
• The iron atoms of hemes typically exist in two
  different oxidation states (2, or 3).
• Oxygen will bind only to the 2 oxidation state.
• In free hemes, there are two open coordination
  sites (on either side of the plane of the
  porphyrin). When oxygen binds to free heme, it
  oxidizes the iron by one electron, resulting in
  superoxide and ferric heme (3 oxidation state).
  The ferric form of heme that is free in solution
  is called hematin.
• This state of the heme prosthetic group is
  inactive, and can no longer bind oxygen.

5
The Role of the Protein Scaffold

• In myoglobin and hemoglobin, the heme cofactor is
  buried deep within a pocket that is composed
  primarily of a-helix secondary structure.
• The heme is held in the protein by numerous
  binding interactions, as well as a covalent
  interaction between the iron and a nitrogen atom
  of a histidine side chain that is located on one
  of the helices.
• The coordination of the histidine to the heme
  completely blocks access of oxygen to this face
  of the heme, forcing it to bind solely at the
  opposite face. This prevents oxidation of the
  heme by oxygen, and allows for simple reversible
  binding.
• The binding of oxygen to the heme alters the
  electronic properties of the heme cofactor, which
  results in different color changes. Heme to
  which oxygen is bound is red in color, while
  ferrous heme containing no oxygen is purplish in
  color (venous blood versus arterial blood).

6
Myoglobin and Hemoglobin
Myoglobin is a compact globular protein. It is a
single polypeptide chain of 153 amino acids,
having a molecular weight of 16,700. It contains
a covalently bound heme group. Myoglobin is the
oxygen storage protein of muscle. It is abundant
in diving mammals like seals and whales, because
they can store oxygen in this form for prolonged
periods underwater. In contrast, Hemoglobin is
a tetramer. Each polypeptide chain is
structurally similar to the polypeptide chain of
myoglobin, and each contains a heme, which like
in myoglobin, is where oxygen binds. The overall
structure of hemoglobin is an a2b2 tetramer. The
a subunit is about 141 amino acids, while the b
subunit is about 146 amino acids.
7
Structural Similarities Between Myoglobin and
Hemoglobin

• Each subunit of hemoglobin has a tertiary fold
  that is similar to myoglobin.
• Myoglobin is composed of eight helical segments
  (shown on the left as cylinders) lettered AH.
  The non helical regions (loops) are labeled with
  the letters of the helices that they connect.
• The histidine that coordinates the heme iron in
  myoglobin is His-93, which stands for the 93rd
  amino acid from the N-terminus of the protein.
  This histidine is also sometimes referred to as
  His F8, which stands for the eigth amino acid in
  helix F.
• Hemoglobin contains 1 heme per subunit, resulting
  in 4 per tetramer.

8
Effect of Protein Structure on Oxygen Binding

• The binding of oxygen to the heme iron is greatly
  influenced by the structure of the protein.
• Notice how there is a cavity in which oxygen
  binds, which is created by valine (located on
  helix E) and phenylalanine (located in the loop
  between helices C and D).
• In free solution, carbon monoxide will bind to
  heme, more than 20,000 times better than oxygen.
  However, when the heme is bound to myoglobin, it
  only binds 200 times better.
• Because of the electronic configuration of
  oxygen, it binds to the heme in a bent
  conformation, and a histidine residue (located on
  helix E) makes a favorable hydrogen bond with it.
• Because of the electronic configuration of carbon
  monoxide, it can bind only in a linear
  conformation. Therefore, the histidine
  sterically hinders its binding to the heme,
  reducing its affinity.
• There is no clear pathway for oxygen binding to
  the heme. Molecular motions (breathing) of the
  protein create transient routes to allow oxygen
  to bind or leave. The rotation of the distal
  histidine is one important molecular motion.

Distal histidine
Proximal histidine
9
Histidine F8 is termed the proximal ligand to the
heme. There is another very important histidine
amino acid in the oxygen binding pocket. His E7
helps to form the binding pocket. Heme will bind
CO 20,000 times more tightly than oxygen, but in
myoglobin, it is only about 200 times. Because
of steric repulsion with HisE7, the binding of CO
is not optimal. It is bent rather than straight
on, which is unfavorable for the electronic
structure of CO. The normal, favorable binding
of oxygen to heme is at an angle. 60 or 120
from the plane of the heme.
10
Effect of Protein Structure on Oxygen Binding

• The binding of oxygen to the heme iron is greatly
  influenced by the structure of the protein.
• Notice how there is a cavity in which oxygen
  binds, which is created by valine (located on
  helix E) and phenylalanine (located in the loop
  between helices C and D).
• In free solution, carbon monoxide will bind to
  heme, more than 20,000 times better than oxygen.
  However, when the heme is bound to myoglobin, it
  only binds 200 times better.
• Because of the electronic configuration of
  oxygen, it binds to the heme in a bent
  conformation, and a histidine residue (located on
  helix E) makes a favorable hydrogen bond with it.
• Because of the electronic configuration of carbon
  monoxide, it can bind only in a linear
  conformation. Therefore, the histidine
  sterically hinders its binding to the heme,
  reducing its affinity.
• There is no clear pathway for oxygen binding to
  the heme. Molecular motions (breathing) of the
  protein create transient routes to allow oxygen
  to bind or leave. The rotation of the distal
  histidine is one important molecular motion.

Distal histidine
Proximal histidine
11
Histidine F8 is termed the proximal ligand to the
heme. There is another very important histidine
amino acid in the oxygen binding pocket. His E7
helps to form the binding pocket. Heme will bind
CO 20,000 times more tightly than oxygen, but in
myoglobin, it is only about 200 times. Because
of steric repulsion with HisE7, the binding of CO
is not optimal. It is bent rather than straight
on, which is unfavorable for the electronic
structure of CO. The normal, favorable binding
of oxygen to heme is at an angle. 60 or 120
from the plane of the heme.
12
Oxygen binding alters myoglobin conformation.
Normally, the iron is not exactly in the plane of
the porphyrin ring. It is displaced 0.055 nm
towards the His proximal ligand. Binding of
oxygen pulls the heme closer into the porphyrin
ring. In turn the histidine is pulled along,
distorting the shape of the a-helix. In
hemoglobin this similar effect on oxygen binding
has profound effects on the ability of other
subunits to bind oxygen. In general, myoglobin
has an intrinsic greater affinity for oxygen than
hemoglobin at all oxygen pressures.
Deoxymyoglobin
13
Protein Ligand Associations

• The functions of myoglobin and hemoglobin are to
  bind oxygen when its concentration or partial
  pressure is relatively high, and then release it
  where it is needed, meaning where its
  concentration or partial pressure is relatively
  low.
• Figure a below represents a hypothetical binding
  curve for some ligand L to some protein. The
  greek letter q, represents the fraction or
  percent of the protein that has the ligand L
  bound to it.
• The curve that is denoted is hyperbolic, and is
  described by the equation y x / (x z). Y
  represents the fraction of ligand bound, x
  represents the ligand concentration, and z
  represents the dissociation constant (Kd) or the
  inverse of the association constant (Ka).
• Notice that when x is very large with respect to
  z, the equation approaches the limit y 1. This
  is the asymptote of the curve. Notice that when x
  is significantly smaller than z, that the
  equation represents a line with slope (1/z).
• Extrapolation to the x axis from the point on the
  curve that represents 50 occupancy gives the
  dissociation constant.

14
Partial Pressure

• The adjacent figure shows that actual curve for
  oxygen binding to myoglobin.
• Notice the nomenclature. The oxygen
  concentration (which is the ligand) is referred
  to as pO2, which means partial pressure.
  Normally, partial pressure is measured in units
  of kilopascals (kPa). Sometimes it is seen to be
  measured in mm Hg or torr (1 mm Hg 1 torr).
• The concentration of a gas or volatile substance
  in solution is proportional to its partial
  pressure in the gas phase above the solution.
• Oxygen accounts for about 21 of the total
  atmospheric mixture of gases. At sea level, the
  total pressure is 1 atmosphere, or 760 mm Hg, or
  100 kPa.
• At standard pressure, myoglobin is fully
  saturated with oxygen.

15
Oxygen Binding to Hemoglobin

• Oxygen binding to hemoglobin is distinctly
  different from its binding to myoglobin. Notice
  that the curve is not hyperbolic it is
  sigmoidal. This is a sign of cooperativity.
• The job of hemoglobin is to bind oxygen
  efficiently in the lungs, where the partial
  pressure of oxyen is about 13.3 kPa however, it
  needs to release it in the tissues, where the
  partial pressure is about 5 kPa.
• Notice how myoglobin is almost fully saturated at
  5 kPa pO2. Therefore, myoglobin would not
  release its bound oxygen to tissues.
• From looking at the sigmoidal curve for
  hemoglobin, it can be seen that it binds oxygen
  relatively weakly at low oxygen concentrations,
  but strongly at high oxygen concentrations.
• This behavior is referred to as allostery.
  Hemoglobin is an allosteric protein.

16
Allostery

• An allosteric protein is one in which the binding
  of a ligand to one site affects the binding
  properties of a ligand to another site on the
  same protein.
• If the same ligand modulates further binding of
  itself to the protein, this is called homotropic
  allostery. If a ligand modulates binding of a
  molecule that is different from itself, this is
  called heterotropic allostery.
• Allostery is frequently observed in multimeric
  proteins, wherein the binding of one ligand to
  one of the subunits, affects the binding of
  ligands to the other subunits.
• Allostery can be positive or it can be negative.
• The sigmoidal curve that is seen can be
  envisioned to be a hybrid curve of two normal
  hyperbolic curves, wherein the dissociation
  constants are different. The high affinity site
  would have a low dissociaiton constant, while the
  low affinity site would have a high dissociation
  constant.

17
Models for Allostery

• Two models for the cooperative binding of ligands
  to proteins with multiple binding sites have been
  advanced.
• The MWC (Monod, Wyman, and Changeux) modelwhich
  is designated the concerted modelassumes that
  each subunit is identical and can exist in two
  different conformations or states. The two
  states have different affinities for the ligand
  however, all subunits within one protein can
  exist in only one of the two states. The binding
  of ligand to a subunit in the low affinity state,
  results in a conformational change that places it
  in the high affinity state. All other subunits,
  even though they do not have a bound ligand, must
  follow suit.
• In the Koshland model, which is the sequential
  model, ligand binding can induce a conformational
  change in just one subunit. This will then make
  a similar change in an adjacent subunit, making
  the binding of a second ligand more likely.

concerted
sequential
18
T and R State of Hemoglobin

• Below are the two major conformations of
  hemoglobin as predicted by the models for
  allosteric activation.
• Oxygen will bind to hemoglobin in either state
  however, it has a signficantly higher affinity
  for hemoglobin in the R state.
• In the absence of oxygen, hemoglobin is more
  stable in the T state, and is therefore the
  predominant form of deoxyhemoglobin. R stands
  for relaxed, while T stands for tense, since this
  is stabilized by a greater number of ion pairs.
• Upon a conformational change from the T state to
  the R state, ion pairs are broken mainly between
  the a1b2 subunits.

19
Subunit Contacts in Hemoglobin

• The strongest interactions in the hemoglobin
  tetramer are between the two subunits that make
  up the protomer unit (a1b1 or a2b2). These
  interactions are shaded in blue in the lower left
  figure.
• The yellow shaded area represents sliding
  contacts. These are contacts made between two
  unlike subunits (a1b2 or a2b1), which undergo
  significant changes upon going from one state to
  the other.
• The a1b1 protomer rotates on top of the a2b2
  protomer about an imaginary pivot in the center
  of the protein, breaking some of the ion pairs in
  the yellow shaded region.

20
The Hill Relationship

• The Hill equation log(q/(1-q)) n L logKd
• Notice that the Hill equation is an equation of a
  line (y mx b), wherein n (the number of
  subunits or binding sites) is the slope.
• The experimentally determined slope does not
  reflect the number of binding sites however. It
  reflects, instead, the degree of cooperativity.
  Because of this, n is usually labeled nH, which
  is called the Hill coefficient.
• For hemoglobin, the hill coefficient is 3,
  although there are four subunits that can bind
  oxygen.
• When there is no cooperativity at all, nH 1.
• The theoretical upper limit for cooperativity
  would be when nH n.
• To adapt the Hill equation to oxygen binding to
  hemoglobin, pO2 should replace L, and P50
  should replace Kd.

21
Heme Structural Changes

• In the T state, the porphyrin is slightly
  puckered. The histidine is not optimally ligated
  to the iron. Because of steric problems, it is
  about 8 from being perpendicular.
• As in myoglobin, the iron lies out of the plane
  of the porphyrin, but moves towards the plane on
  oxygen binding.
• The subsequent distortion of the helix to which
  His F8 is bound, necessitates that the subunits
  adjust their conformations.
• These changes lead to adjusments in the ion pairs
  at the a1b2 interface.

22
Heterotropic Cooperativity in Hemoglobin

• Exam on Thursday March 6, 2003.
• Exam will cover everything up through Hemoglobin,
  including the hemoglobin portion of todays
  lecture. The exam will not cover immunoglobulins
  or actin and myosin.
• People with last names beginning with AC or
  asked to report to room 016 Ag Sci for the exam
  (same room as for the previous exam).
• So far we have talked mainly about homotropic
  allostery, wherein a particular molecule can
  influence the binding properties of other like
  molecules.
• In heterotropic allostery, a particular molecule
  can influence the binding of molecules that are
  unlike it in structure.
• Note that hemoglobin has a number of different
  functions.
• Protons, carbon dioxide, chloride ions, and the
  mtabolite 2,3-bis-phosphoglycerate all affect the
  binding of O2 by hemoglobin.
• Remember that carbon dioxide is a byproduct of
  cellular respiration, and must be removed from
  the tissues.

23
Oxygen-dependent metabolic processes in tissues
result in CO2 formation as a byproduct. The CO2
must be transferred from the tissues back to the
alveoli of the lungs, so that it can be
effectively expelled. In return, oxygen must be
transferred to the tissues.
pO2
Y
pO2 P50
24
Proton uptake by hemoglobin in tissues is
mediated by the relatively high concentration of
CO2. Remember the hydration of CO2 by
the zinc-dependent enzyme carbonic anhydrase.
25
The Bohr Effect

• Proton uptake by oxyhemoglobin promotes
  dissociation of oxygen.
• At lower pH values, the oxygen binding curve
  would be displaced towards the right. This means
  that the partial pressure of oxygen required to
  give 1/2 maximal binding would be increased.
• When sigmoidal curves are displaced towards the
  right, this signals greater cooperativity or
  allostery.
• Protons are antagonists of oxygen binding. They
  suppress the binding of oxygen.
• Protons are negative heterotropic allosteric
  effectors.

HbO2 H HbH O2
26
How Are Protons (H) Exported?

• Oxygen and protons do not bind to the same site
  in hemoglobin.
• Protons bind to several different amino acid
  residues in the protein.
• One amino acid side chain that makes a major
  contribution to the Bohr effect is Histidine 146,
  which is located on the b subunits.
• When protonated, Histidine 146 makes an ion pair
  with aspartate 94. This ion pair is one of those
  that helps to stabilize deoxyhemoglobin in the T
  state. Remember that oxygen binding to
  hemoglobin in the R state occurs with a greater
  affinity (lower Kd).
• In the T state, the ability to form the
  electrostatic interaction drives the pKa of
  Histidine 146 up.
• In the R state, in which the ion pair cannot
  form, Histidine 146 has a normal value of 6.0.
  At pH 7.6, which is the pH of blood in the lungs,
  this residue is largely unprotonated.
• As the pH falls, protonatation of Histidine 146
  causes a transition to the T state, and causes
  the other b-subunit to pick up a proton, because
  its pKa is driven upwards.
• Other sites of proton binding include the
  amino-terminal residues of a subunits, a few
  other histidine residues, and a couple of other
  amino acids.

27
How is CO2 Exported?

• Although dissolved carbon dioxide will cause the
  medium to become acidic, which is attributable to
  its hydration to carbonic acid, and subsequent
  proton loss, another reason for forming carbonic
  acid is to solubilize CO2, which is a very
  nonpolar molecule. The bicarbonate is
  transported to the lungs where the partial
  pressure of oxygen is higher. Upon oxygen
  binding to hemoglobin, protons are release, which
  combine with bicarbonate to form H2CO3. Remember
  again that H2CO3 and CO2 are in equilibrium, and
  the CO2 can then be released.
• Carbon dioxide is transported in the form of a
  carbamate on the amino terminal residues of each
  of the polypeptide subunits. Note that the
  formation of a carbamate also results in release
  of a proton into solution. Therefore direct
  binding of carbon dioxide to hemoglobin also
  indirectly induces the Bohr effect.

28
2,3-Bisphosphoglycerate

• 2,3-Bisphosphoglycerate is an important
  allosteric effector of hemoglobin. It is present
  in erythrocytes at about 5 mM (at sea level),
  which is a fairly high concentration (about
  equivalent to tetrameric hemoglobin). At high
  altitudes it is present at 8 mM. At high
  altitiudes, wherein the partial pressure of
  oxygen is low, one would want hemoglobin to give
  up more of its bound oxygen to the tissues.
• One molecule binds at the interface of all four
  subunits, and makes contacts with the b-subunits.
  Its binding stabilizes the deoxyhemoglobin
  state. This promotes oxygen dissociation from
  oxyhemoglobin.
• 2,3-Bisphosphoglycerate (BPG) is another negative
  heterotropic effector.

29
2,3-bisphosphoglycerate Binding to Hemoglobin
The negative charges on 2,3-bisphosphoglycerate
interact with positive charges on hemoglobin
(shown in blue)
Shown here is the R state of hemoglobin, to which
oxygen has a greater affinity. Notice how the
binding site for BPG collapses.
2,3-bisphosphoglycerate binding to hemoglobin
stabilizes the T state.
30
Sickle Cell Anemia

• Sickle cell anemia was the first condition for
  which a genetic mutation was correlated with a
  physiological response. This is a homozygous
  recessive condition, in which offspring must
  inherit both of the mutated genes in order to
  develop the disease fully.
• There are more than 300 different genetic
  variants of hemoglobin that are known.
• In the case of sickle cell disease, a valine
  residue is substituted for a glutamate residue in
  the b chains. This results in two fewer negative
  charges for the tetrameric structure.
• The substitution of a hydrophobic amino acid for
  a hydrophilic one makes the resulting molecule
  sticky. This is because a hydrophobic patch
  has been created, which causes molecules to stick
  together at this point. This causes aggregation
  to occur in deoxyhemoglobin.
• Subsequent to strand formation, several strands
  can assemble to form an insoluble fiber, which is
  what gives sickled cells there shape.
• People with sickle cell anemia suffer from
  repeated crises brought on by physical exertion.
  The hemoglobin content of their blood is about
  1/2 of normal erythrocytes, and the sickled cells
  can block capillaries, causing severe pain.