Download 8/20/08 Transcript I - UAB School of Optometry

FUNdamentals 1
10:00 – 11:00 8/20/08
You’ve had an introduction to information high technology. We will go to “real,
valuable scientific information.” Today we are going to talk about protein dynamics and
using hemoglobin as an example. We will also touch on protein dynamics Friday, when
we talk about protein folding because this is where dynamism of a protein is most
obvious. Today we are going to get into muscle as well, so tomorrow we can devote a lot
of time to the collagen, which is relevant to the principles of dentistry and optometry.
Friday, we will finish up the protein folding. Today we are going to pass general
chemistry of proteins and launch into some specifics (e.g. hemoglobin, muscle, collagens,
protein folding, and energetics of folding). Changing directions today.
Note: slide from yesterday that was missing is in the section entitled “The Collagens”
Things you should know by now:
 Definition of a globular protein.
o What constitutes it?
 How beta-pleated sheets are used in a differential manner.
o Two kinds exist:
 one has hydrophobic side chains on both side of sheet
 the other kind is amphipathic (one type of side chain on one side of
the sheet, and another on the other side)
 Myoglobin structure (overall)
 General overview of fibrous proteins and examples
 What is necessary to have a coiled-coil?
o You have to have some type of repeat.
o Two general types of repeats are a heptad repeat with seven amino acids,
and another one is a tri-repeat with three amino acid repeat that you find in
collagen
 Quaternary structure (advantages)
 Be able to identify some proteins which actually have a quaternary structure
Slide 1
 We use hemoglobin as an example of a protein, which actually moves when it
functions. It moves over and above the movement you would have on the part of a
protein simply from Brownian motion.
o Basic phenomena here is a molecule that is going to go from the deoxystate (no oxygen) to the oxy-state (when it has taken up oxygen), and
become a different kind of molecule now.
 This is a process that can be looked upon just like a chemical reaction.
o You take hemoglobin in the deoxy-form, and expose it to oxygen and it
will take up form.
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o On the other hand, if you take the oxy-form of hemoglobin and you
transfer it to an area where there is low oxygen tension, it will transform
itself back to the deoxy form. It is simply a chemical reaction.
A protein quaternary structure evolved for the transport of O2 and CO2 as well as
protons between tissues and lungs.
o Also transports nitric oxide molecules.
It is basically a transport type of molecule and it designed to be that way
Transport requires (for oxygen) a prosthetic group, the heme moiety carrying a
ferrous atom.
o It cannot be a ferric ion, because it will not complex with oxygen properly.
It has to be in the reduced form of iron—ferrous iron.
Slide 2
 Myoglobin is a protein of about 153 amino acids.
 The two types of structures that are involved in hemoglobin are:
o Alpha 1 and Alpha 2 (identical)
o Beta 1 and Beta 2 (identical)
o Main point: there are two types of chains in a hemoglobin molecule.
 A hemoglobin molecule is composed of these four (distinct) subunits, each of
which has a structure similar to myoglobin. So similar that the heme molecule fits
into a pocket into each of the subunits of the hemoglobin molecule.
 A hemoglobin molecule has 4 subunits, 4 poryphrin rings, and 4 ferrous iron
atoms inside each ring.
 When we come to quaternary structure for collagens, there are not set number of
molecules or subunits.
 Myoglobin has only one kind of chain.
 Hemoglobin has two kinds of chains.
 It takes two genes to operate to make a hemoglobin molecule.
 One different gene to make the myoglobin molecule.
 The subunits that come together in this fashion to make hemoglobin and
myoglobin remains separate. By now you should understand a rational for that.
Very similar molecules, similar primary, secondary and tertiary structures. One
type of molecule (one set of polypeptides) goes into a quaternary structure. The
other one remains independent and functions independently forever more. Why
would you think that occurs?
o Answer: Myoglobin has less hydrophobic external regions, so it doesn’t
need to bind to other proteins.
o Myoglobin has hydrophilic amino acids on its surface. It loves the water.
It will not associate into a quaternary structure.
o The alpha and beta chains of hemoglobin have more hydrophobic bonds
on their surfaces and they are coming together to hide those hydrophobic
surfaces. In doing so, they generate a quaternary structure, which is
definitive. The position of the hydrophobic side chains, dictate how these
subunits are going to come together and act as a single type of molecule.
Slide 3
 You can tell how this situation is arranged because the general features of
hemoglobin have already been discussed.
 The heme resides in the hydrophobic pocket, which excludes water. It keeps the
heme molecule because it is a hydrophobic pocket, and the heme molecule is
hydrophobic.
 It prevents the heme iron from causing dimerization.
 It ensures that all ligands less than at a maximum angle (will be discussed later).
 This is generally why you have to carry heme around in a hydrophobic pocket.
 Remember yesterday when I discussed the side view of a heme molecule? This is
a flat molecule. You are looking now at the surfaces, and the iron atom.
 If it were not for the hydrophobic pocket, the iron could be complexed with
oxygen and the oxygen could give rise to dimerization of heme molecules.
o Water would also react with the iron and cause it to be oxidized to ferric
iron. There are many problems with that.
o (Side note) Considering that, think about this…there were people at one
time who were looking for the Holy Grail, and the Holy Grail here is a
substance that you can add to the blood and resupply oxygen transport
systems. In other words, if you lost a lot of blood, you have to have a
blood transfusion. It has to be checked out that the blood that you receive
is compatible with your blood type. The blood could potentially carry a lot
of diseases…you are at the mercy of that transfusion system. The idea is to
generate a blood transporting system that could be infused off the shelf. In
other words, you don’t have to worry about immunology, disease, viruses,
etc. One notion was to just transfer/infuse heme with the iron into a blood
stream and that should do the job. Obviously that never worked.
o There has been success in infusing a modified hemoglobin that carries
heme though recently. This would be great because you would have the
ability to replenish blood supply and oxygen transport capability without
worrying about any kind of biological problem, it would be a simple
chemical problem.
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Globins are united noncovalently in a tetrahedral array.
The way you know that is because there are 2 alpha chains and 2 beta chains in
the adult hemoglobin, the contacts are alpha subunit, beta subunit, beta to alpha,
alpha to beta. That is, there are no type alpha-alpha and beta-beta contacts. There
are contacts among the four, but they are always with the opposite.
If you dilute hemoglobin, it will dissociate into particles (alpha and beta; alpha
beta). The tetramer dissociates into two dimers.
o In order to separate the tightly connected dimers, you have to denature the
protein by warming it to a point where its tertiary structure can no longer
be maintained and its secondary structure is destroyed and only the
primary structure remains (the random coil before folding).
Slide 4
 How hemoglobin and the position of the heme molecule actually protects us…
 If you have free heme, and you are treated it with carbon monoxide, which is a
poison to the body by taking the place of oxygen on the hemoglobin molecule and
attaching itself to iron, and you free heme, carbon monoxide can react with the
iron atom and give a straight-line configuration with the bonding in that situation.
o In this situation, it is virtually impossible to remove carbon monoxide.
o The heme molecule is in the pocket, and carbon monoxide has to come in
and be associated with the iron atom in an angular way.
o It is done because on the opposite side, where oxygen and carbon
monoxide come in, there is another histadine residue that prevents carbon
monoxide from binding tightly. In this case, carbon monoxide can be
removed. Oxygen comes in that way as well, and if it were not for this,
oxygen would bind tightly and never leave.
o The transport phenomena of hemoglobin would be compromised severely
if all of these agents (carbon monoxide and oxygen) were attaching in a
tight fashion.
 They have to attach in a loose fashion, because the histadine is
holding the iron in the poryphrin ring.
 The histadine from the f-helix (f8 because 8th amino acid on the f
helix) and the histadine on the other side is histadine E7, which is
the 7th amino acid in the E-helix play a large role here.
 The E & F helices are very important in this structure.
 The way to get over carbon monoxide poisoning is by infusing the
system with large quantities of pure oxygen.
 In this situation, oxygen can compete effectively with
carbon dioxide and eventually return to normal oxygen
transport. There is a way to get rid of carbon monoxide.
However, if it were not for this angular bonding, carbon
monoxide would be there forever, as well as oxygen and
not be able to be delivered to the tissues. This is an
example of how the tertiary structure of the hemoglobin
subunits work in concert with a prosthetic group to give
you a great deal of activity, which is beneficial to the
organism.
Slide 5
 Actual functioning of a hemoglobin molecule now…
 Looking at hemoglobin in contrast to myoglobin here
 Diagram is of oxygen intake: hemoglobin (purple line), myoglobin (blue line)
 Zero torr, to 100 torr (atmospheric pressure)
 In this case, myoglobin is exposed to oxygen and takes it up rapidly and will go
up to a level of 100% saturation quickly.
 Hemoglobin is reluctant to take up oxygen. It will not enter into an oxygen-uptake
process that is linear (quick), until you reach about 20 torr. It will soon reach
100% saturation at 100 torr.
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In the internal area of your body, outside of the lungs is where the oxygen partial
pressure is low (about 26 - 30torr). At that point, hemoglobin reverses from the
oxygenated hemoglobin (from the lungs) and now travels to tissues and gives up
its oxygen to myoglobin (precisely what it is supposed to do).
o On one pass through the tissue, note that hemoglobin gives up about 25 –
30% of its oxygen. It doesn’t give up all the oxygen and go to zero; there
is a safety mechanism that prevents it from giving up everything because
you don’t want to have every pass of hemoglobin give up its oxygen at
rest, because you would soon be depleted of oxygen.
o During periods of little activity, hemoglobin will pass through the tissues
still retaining about 70% of its oxygen carrying capacity and giving up
about 30%.
How does this happen? Why does this happen? Think back to what we talked
about yesterday, when oxygen comes to bind to the iron atom in the poryphrin
ring. When that happens, the iron atom moves to the other side of the poryphrin
ring, causing the f-helix to move and cause all the other helices to shift position.
Myoglobin can do that very readily because it is a “stand alone molecule,” there is
nothing to prevent its helices from shifting.
o In hemoglobin (tetramer shape) one movement of the f-helix and one
subunit will have to move and cause all others to move as well.
o Its much more difficult for hemoglobin to take up oxygen, than it is for
myoglobin. You can see this all the way along the pathway here.
o Hemoglobin really becomes fully oxygenated only in the lungs. Outside of
the lungs, it is open to deliver its cargo.
Slide 6
 Example of the histadine from the f-helix holding the iron atom in this particular
position, on the upper surface of the hydrophobic heme molecule in this diagram.
 Oxygen comes in there and changes the whole aspect. The f-helix is going to have
to move. Oxygen has moved the iron atom, which is not depicted here.
o What you should really concentrate on here is the movement of the fhelix, the histadine side chain, and the iron atom that has moved in.
o That is what drives this whole process—the movement of the polypeptide
chains…dynamics of proteins!
 The necessity to move around also brings in the concept of fragility; it makes
proteins a little fragile.
o Every hemoglobin molecule that you have today will not be around four
months for now. Hemoglobin molecules change and will turn over
constantly. About a 90 – 120 day life of a hemoglobin molecule.
Slide 7
 See above notes…similar diagrams.
Slide 8
 This describes what I just told you. When the first oxygen binds to the iron in the
heme of the hemoglobin, the heme iron is drawn into the plane of the poryphrin
ring.
o This binding is slow because it initiates a series of conformation changes,
which has to be transduced into all the other helices in the quaternary
complex.
o The more oxygen that is bound, the more likely there is oxygen to be
bound, this is called positive cooperatively. In other words, oxygen is a
positive oxygenation factor for hemoglobin.
 Making crystals of hemoglobin molecules, then you expose that crystal to oxygen,
then the crystal shatters because the molecules have to move. The crystal structure
is obliterated. That is a good example of how the molecules (peptides) have to
move when confronted with oxygen.
Slide 9
 This diagram is not particularly informative.
 This is deoxyhemoglobin and the oxygenated form. This is a moving picture of
hemoglobin as oxygen is being taken up.
 A particular part moves to the left, so you have about a 15-degree shift of the
position of beta and alpha, relative to the beta and alpha behind the plane.
 When the fully oxygenated hemoglobin is present, you will see this shift.
 The two subunits in the front, relative to the two subunits in the back are the focus
here. This just gives you an idea of the kind of shifting that must occur during that
process.
Slide 10
 Again, we have a problem of taking up oxygen until you get fully saturated in the
lungs, but then that problem of taking up oxygen is put to good value in the
reverse where the hemoglobin will transfer the oxygen to the myoglobin. The
myoglobin will take up the oxygen to be utilized in aerobic metabolism.
Slide 11
 This is a complicated diagram. (Note: Dr. Miller moved around a lot on this
diagram so the following notes may be a little confusing taken out of context. Just
refer back to diagram throughout this section.)
 This illustrates the kinds of bonds that are available within the quaternary
structure. If you look at the C-terminal part of the beta chain, you will have a
carboxyl group and it can bind with the lysine side chain.
o Ionic bond between alpha and beta chains.
o Aspartic acid on the beta chain that is close to a histadine can result in an
ionic bond there, which is very important with respect to one of the effects
we will discuss in a minute.
 Remember the aspartic acid close to a histadine on a beta chain.
 This is the same thing (pointing on slide) only in the opposite direction,
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o C-terminus of the beta chain making a carboxyl group negatively charged
interacting with lysine.
o Notice the amino group acting with the carboxyl group on the edge the
amino terminus of the alpha chain and the carboxyl chain of the alpha
chain.
o Notice arginine/aspartic acid interactions.
o There are all kinds of interactions within a structure such as hemoglobin.
o Main point: these interactions have to be broken when the molecules shift.
When the components shift, a lot of these interactions have to be broken
and that is the forced that has to be exerted to change the conformation has
to break many of these bonds and reform them.
It is not really important that you grasp all of the interactions in this slide because
it would take us a whole semester. This is just an example.
Slide 12
 The Bohr Effect describes the competition between oxygen and hydrogen ions
(protons).
 Binding of protons diminishes oxygen binding. This occurs down in the tissues
where there are a lot of protons (acid), because of metabolic activity.
o At this particular point, you want to deliver as much oxygen as possible.
You need a little help here and this comes from the fact that in the tissues
there is a great deal of hydrogen ions (low pH).
 Also, the binding of oxygen diminishes proton binding.
 Resulting equation of these two concepts:
o oxyhemoglobin + protons = protonated hemoglobin + free oxygen.
HbO2 + H+ <> HbH + O2
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This is precisely the type of reaction you would like to have occurring in the
tissues. The tissues, being metabolically active, will produce a lot of acid
(characteristic of our metabolism) The acid will exert itself in the form of protons,
and the protons compete with oxygen and will give the oxygen an extra boost to
get off and be delivered in the proper location.
Similar considerations apply to CO2 due to the dissociation of H2CO3 (carbonic
acid). CO2 will allow carbonic acid to be formed and will also give rise to
protons.
o CO3 can act with the amino terminus of some of the chains in the
hemoglobin molecule and will carbamylate the chains. We won’t go into
detail of this yet.
o Focus on the protons being delivered directly form metabolic activity of
the production of CO2 & CO2 giving rise to protons when carbonic acid
dissociates. This situation is to our benefit.
Slide 13
 The myoglobin reactivity with oxygen and hemoglobin at normal pH (green).
o As the pH is lowered and the hydrogen ion concentration is greater, then
more and more oxygen is delivered.
o For example, a of pH 6.8 will give you a greater transport (greater
unloading) of each passage through the venous tissues. You go from
normal pH of 7.4 where about 75% of oxygen remains with the
hemoglobin down to a pH of 6.8 where on every pass only about 50% of
the oxygen remains.
o The system is set up for our benefit. The more active you are, the greater
the need for oxygen and the greater amount of oxygen is delivered on each
passage through the tissues. The Bohr Effect is beautiful system for
complimenting the normal function of hemoglobin.
 The pH does not affect myoglobin. There is a chemical reason for this that will be
discussed later.
Slide 14
 Another situation arises, that is we have another negative effector for oxygen
transport—2,3 Bisphosphoglycerate.
o This is a highly anionic substance.
o It binds to hemoglobin, when hemoglobin starts to be deoxygenated.
o As you start to go into the deoxy-state, the central cavity between the
hemoglobin subunits gets larger. The previous slide of the shifts of the
chains relative to each other (slide 9) didn’t show this, but the cavity gets
larger as the molecule loses oxygen and goes into the deoxy state.
 When this occurs, 2,3 Bisphosphoglycerate—will enter that cavity
and promote deoxygenation. In other words, that cavity is enlarged
and is kept enlarged when 2,3 Bisphosphoglycerate.
 As long as you get that cavity enlarged, you are promoting deoxy
state. You are promoting the deoxy state when:
1.) protons are present
2.) 2,3 Bisphosphoglycerate are present
Slide 15 - 2,3 Bisphosphate structure
 3 carbon chain of glyceric acid is present here
 If all three of the carbons had hydroxyl groups, this would be glycerol, but this is
glyceric acid (the acid form of glycerol)
 there is a phosphate group esterified to each 2,3 carbon atoms (hence the name)
 “bis” – meaning phosphorylated twice