Recent Studies of the Structure of Proteins
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00:00
Chemical structureProteinNobeliumSemioticsChemistryOrigin of replicationDelicatessenUreaWine tasting descriptorsHelixHardnessMeatMoleculeHemoglobinKernproteineEnzymeErdrutschSpeciesMessenger RNALactitolAtomic numberSystemic therapyBiomolecular structureHorse meatMolecularityCommon landBerylliumProteinChain (unit)TiermodellDNS-SyntheseQuartzChemical structureActivity (UML)Multiprotein complexChromosomeAlpha particleProcess (computing)AmineChemical reactionGlassesRibosomeCobaltoxideFunctional groupChemistryDenaturation (biochemistry)PeptideWursthülleSpontaneous combustionMaskierung <Chemie>HelixWaterPharmacyAmino acidCell (biology)Peptide sequenceScreening (medicine)Residue (chemistry)Combine harvesterGas cylinderWhitewaterDensityWasserbeständigkeitSetzen <Verfahrenstechnik>AtomGrowth mediumSide chainConnective tissueGolgi apparatusPeptideIronHistidineElectronic cigaretteNucleic acidPhysical chemistrySpeciationPolymerTheoretische ChemieMetallmatrix-VerbundwerkstoffPressurePH indicatorSet (abstract data type)ProteinfaltungStorage tankWine tasting descriptorsPolystyreneChemical propertyDeterrence (legal)RNASecretionX-ray crystallographyReflexionsspektrumCalculus (medicine)Red blood cellArtificial leatherChemical bondHope, ArkansasBeta sheetPaste (rheology)ThermoformingConcretionWattAreaWaterfallPilot experimentMatchAusgangsgesteinCell growthLibrary (computing)Starvation responseKristallkörperPeriodateAcidHydrogenHyperpolarisierungHost (biology)Carbon (fiber)FoodButcherMan pageSpring (hydrology)GesundheitsstörungReaction mechanismFireChemical compoundAgeingPhenylalanineColourantPatentRiver sourceLecture/Conference
Transcript: English(auto-generated)
00:15
Ladies and gentlemen, I feel very happy that even if I was late for all the other lectures,
00:25
at least I arrived in time for my own. And I'm happy also to be here in Lindau. I feel like what we call in Cambridge a freshman, a first year student of Lindau,
00:42
and this is a very pleasant experience. Now, I want today to talk a little bit about the structure of proteins. Those of us who read the Scientific American and other publications of this kind
01:04
are very familiar nowadays with the work which has gone on in the last few years on this other very important biological molecule, DNA, which means deoxyribonucleic acid,
01:25
or if one is writing in German, one should say DNS, deoxyribonuclein soda, and that this molecule in the chromosome of the living cell and in the nucleus
01:49
carries the hereditary information which comes out from the nucleus in the form of another related molecule which is called messenger RNA,
02:08
or ribonucleic acid, and this molecule attaches itself to the so-called ribosomes, and there it makes protein.
02:23
Now, we can look at some details of this process. The first slide, please. Here is a picture of a chromosome, and in the chromosome is the DNA, and you can see where in the chromosome there is a swelling here,
02:48
and the swelling is a part of the chromosome which is at this moment active. That part of the chromosome is making the messenger RNA.
03:00
The next slide, please. Here we have the DNA in the nucleus making the messenger. The messenger comes out and attaches itself to a ribosome, and in the next slide we can see here the messenger RNA,
03:21
and here are the ribosomes attached to it. Now, what is all this for? The messenger RNA ribosome complex is making a protein molecule. The protein molecule is a long chain of amino acids,
03:45
and the sequence, the order of the amino acids along the chain is determined by the so-called code, the code of the nucleic acid transferred from DNA to RNA
04:04
and then converted into protein. Now, this has been the important advance in our knowledge in the last few years of the way in which the DNA, the hereditary material, controls the activity of living cells.
04:26
And I want to talk to you from this point on. What happens next? The apparatus here makes this long chain, and later this long chain is folded up into a more spherical shape.
04:47
We have the long protein chain, the polypeptide chain.
05:02
It is made of these single units. The single units are called amino acids, and there are about 20 types of amino acids. The whole chain might be 100 or 200 of them.
05:27
Now, this is how the polypeptide chain is built, but when the protein is in action in the cell, it is not like this. It is folded up into some kind of nearly spherical shape,
05:43
and we know that the most important kinds of proteins in the cell are the enzymes, the enzymes which have the function of carrying out the chemical reactions of the living cell,
06:01
of converting some substance A into another substance B. This is a specific catalytic reaction of the enzyme. And what we shall be interested in today is what is the relation between this polypeptide chain,
06:22
which in some way folds itself up and converts itself into the enzyme or other protein molecule, and then how it performs its function in the cell. Now, this spherical structure of the protein molecule in the cell
06:46
is a highly specific one. You can take a long chain like this. If you fold it up, there are many different ways in which such a folding would be possible. But in the living cell, only one way is chosen.
07:04
The folding is a highly specific one. We can see this from the fact that each enzyme performs just one specific function. It has a special arrangement of the amino acids in the chain
07:22
specially designed to perform that function alone, and this demands a highly specific arrangement. Another piece of evidence we have of this specific arrangement of the protein is the fact that many proteins will form crystals.
07:41
The next slide, please. In the next slide, I have some crystals of a protein molecule. This is the protein myoglobin. And the fact that the protein makes crystals like this means that every molecule must be the same as every other molecule.
08:03
You can only form regular crystals, which of course are regular arrangements of molecules. You can only do this if the molecules are very similar to one another. Now, the fact that we get crystals from some protein molecules
08:23
gives us an opportunity to study their structure using the methods of X-ray crystallography. And, in fact, this is the technique with which I and my colleagues have been studying these structures.
08:41
Today, I shall not discuss at all the X-ray methods. I think perhaps you will be more interested not so much in the methods themselves as in the results. Now, we want to study from these crystals
09:00
to study what is the structure of a protein molecule. We would like to know this, first of all, because simply of the intrinsic interest of examining a very complicated molecule, seeing what it is like, and I must remind you that a protein molecule
09:22
may have in its chain several hundred amino acids. This means that the whole molecule contains several thousand atoms and some proteins are very much bigger than that, so they are extremely complicated things.
09:42
If we understand about the structure of the molecule, this may help us to get some information about the other problems I've already mentioned. First of all, the problem, how is this molecule made? How is it manufactured?
10:02
In other words, how does this process of folding up the long chain into the spherical molecule, how does this happen? And secondly, if we understand the structure, we can perhaps learn something about the specific action
10:22
of the protein molecule, its function as an enzyme, or used for other purposes. We can understand that if we know something about the structure. And I shall talk entirely today about two proteins
10:44
with which I have been particularly associated, the protein hemoglobin and myoglobin. Now, just to say a little bit about, to tell you a little bit about these molecules,
11:05
hemoglobin is the protein in the red blood cells, the protein which has the function of carrying oxygen from the lungs to the tissues in the bodies of animals.
11:23
It's an oxygen carrier, and it is a fairly middle-sized protein. It has got something like 10,000 atoms in it,
11:41
and those 10,000 atoms are composed of four polypeptide chains, and each chain carries a heme group. Now, we can look in the next slide at a picture of the heme group.
12:05
This is a flat group of atoms with an iron atom in the center, and it is this iron atom which carries an oxygen molecule. And hemoglobin has four heme groups,
12:23
so it contains four iron atoms, carries four oxygen molecules. That is one of the two proteins that I shall be concerned with, and this is the protein which my colleague Max Perutz has been studying for many years.
12:43
I myself have studied a simpler protein, one which is perhaps less familiar to you, the protein myoglobin, which is also an oxygen-carrying molecule,
13:01
but it is not in the blood. It is contained in the cells of the muscles of the tissues of the body, and the way it works is that the hemoglobin brings the oxygen to the tissues and passes it on to the myoglobin, and the myoglobin acts as a store of oxygen until the oxygen is needed in the cell.
13:28
Now, I chose myoglobin because it is a simpler protein. It is one of the simplest proteins we know. Still you see rather complicated 2,500 atoms,
13:43
but less complicated than hemoglobin, and whereas hemoglobin has four polypeptide chains, myoglobin has one chain, it has one heme group, and so it can carry one oxygen molecule.
14:04
And this is the protein I have been studying for a number of years, and I want to try to tell you something about its structure so that from this basis we can hope to understand a little about some of the other problems.
14:26
Now, I told you that I have no intention of talking to you about X-ray crystallography, but let us look in the next slide at an X-ray photograph.
14:41
This is an X-ray photograph of the crystals which I showed you a few minutes ago, and the problem of the X-ray crystallographer is to study photographs like this and from these to deduce what is the structure of the molecules in the crystals
15:03
which produces this pattern of spots. Now, you see that it is very complicated. Indeed, it is much more complicated than this because here we do not have the whole X-ray pattern of a protein crystal, only part of it.
15:21
In fact, the X-ray pattern of myoglobin crystals has perhaps 25,000 spots in it. One studies a structure like this in stages. It turns out one can begin with the spots near the middle of the pattern.
15:41
One gets what we call a low-resolution picture of the molecule. One then brings more and more reflections into the calculations, and so the pattern becomes sharper and sharper. The next slide, please.
16:02
It is as if we are looking at a molecule like this. You see, this is a ring of atoms, and here we are looking at it rather sharp, but supposing at the beginning we had a very bad pair of spectacles,
16:24
we would see just a solid piece of density here, and as we gradually improve, we put on better and better spectacles, we get a sharper and sharper picture, and in the protein work we begin with a low-resolution picture,
16:46
taking only a few reflections into account, and by degrees we try and make the picture sharper and sharper until eventually we hope to see every atom in the structure.
17:02
Now, in the next slide is a picture of our first map of the myoglobin molecule. The way we make a model like this, you have to imagine here is this molecule.
17:21
It is in three dimensions. It is rather difficult to represent this because our screen is only two dimensions, so what we do is we take the molecule and we cut a number of parallel sections through it, and we put the density of atoms in each section onto a set of transparent sheets,
17:48
and we pile them on top of one another, and so we get a three-dimensional representation. And here the spectacles with which we look at the molecule are not very good ones.
18:02
The most that we can see here is some indication of this long polypeptide chain going around through the molecule. You can see a little bit of it there. We can also see the iron atom.
18:20
I told you that myoglobin contains a single iron atom. The iron atom is very dense, much more dense than any other atom in the molecule, so we can see that iron atom as a very dense peak. And by looking at a map like this, we can arrive at a model of the myoglobin molecule.
18:48
The next slide, please. Here is a model of the myoglobin molecule, again looked at not sharply. We do not see the individual atoms,
19:02
but we could see the way in which this polypeptide chain goes around the molecule, and here is the iron atom and the heme group. You will see it is a very irregular, a very complicated object,
19:21
and it was quite surprising when we first saw this to think that this thing could be so irregular in its arrangement. Well, later we put more X-ray reflections into our calculations
19:43
and got a better map of the structure. At this resolution, we see the polypeptide chain simply as a solid, cylindrical arrangement. We do not see its individual atoms.
20:02
We cannot tell how the atoms in the chain are arranged. The next slide, next. Here we have the next map of the myoglobin molecule, and we were very happy when we saw this map,
20:20
because what had been in the old map a solid cylinder of density for the polypeptide chain, we now saw that this cylinder, here we are looking at the cylinder end-on, we saw that it is hollow, there is a hole down the middle.
20:42
And this made us realize that, as in so many things, our colleague, Dr. Pauling, had been right when 10 years before we obtained this map, or nearly 10 years before, he had proposed a structure for the polypeptide chain.
21:03
The next slide, please. The so-called alpha helix, a spiral arrangement of the chain, and you see, being a spiral, like a spring, it means that if you look down the middle, you can see a hole.
21:23
We were able to see this hole in the map. And by looking at this map, we began to be able to construct models of the molecule, putting in some of the individual atoms.
21:42
In the next slide is one of our more recent maps of the myoglobin molecule, and here you can see this is where all the individual atoms are, and with this kind of map, one can really begin to understand the chemistry of the molecule.
22:03
Those of you who are biochemists will perhaps recognize, here we have the heme group and the iron atom, the heme group edge-on, and it is attached to the rest of the molecule by a group here,
22:21
which you can see has got five atoms in a ring, and the biochemists will immediately recognize this as the amino acid, which is called histidine. And, of course, many years before we saw this histidine in the model,
22:41
the biochemists had imagined, for various reasons which I have no time to discuss, they had imagined that this connection was histidine, and this turns out to be correct. So, we have many maps like this, and we try from these maps to construct a model of the molecule in which we put in all the atoms.
23:08
There are 2,500 atoms. The model is rather complicated. Here is a model of the myoglobin molecule.
23:22
I can assure you there are 2,500 atoms there. The white ones are hydrogen, the black ones are carbon, the red ones are oxygen, and so on. And the only difficulty about this model is that you can learn nothing about how this molecule is constructed.
23:48
It is a solid thing. You cannot see what is inside it. And even if I had the actual model here, instead of simply a photograph, it would still be very difficult for you to understand the construction of the molecule.
24:06
Here is another model of the molecule in which we have, so to speak, taken the flesh from the bones, we have stripped away the atoms, we have simply left the connections between them,
24:26
and the polypeptide chain has been marked here with a white string, and you can follow it all the way around the molecule. And every time the string goes straight like this, one has got an alpha helix,
24:47
one has got one of these spiral arrangements of the chain. It turns out that something like 70% or 75% of the polypeptide chain in the molecule
25:02
is made up of these straight segments of helix. Here is a still more simplified version of the same model. We start here at the end of the chain, and you see here we have a spiral arrangement for a time,
25:23
and then we get a little bit irregular at the corner, and another spiral, and so on. One can follow it all the way around, and there is the iron atom, the iron atom which attaches the oxygen molecule, and the heme group, which is attached to the rest of the protein by means of this histidine residue here.
25:50
Well, as I told you, my difficulty is that this molecule is a very complicated one. It is three-dimensional, and unfortunately our screen here is only two-dimensional.
26:07
It is quite difficult to give you a good idea of the way in which it is constructed. But one might ask, first of all, what are the forces which hold together this molecule
26:24
into a spherical shape, or nearly spherical shape? How does this piece of chain, how is it attached to its neighbours? Now, there are no, in some proteins, there are actual chemical bonds
26:45
between one piece of chain and another piece of chain. This is not the case in my robin. There are no chemical bonds between neighbouring pieces of chain. So, we ask, what are the forces responsible for maintaining the integrity of the structure?
27:08
For a number of years, before any precise picture of a protein molecule had been obtained, for a number of years, the physical chemists have been thinking about these problems,
27:21
and they noticed that if you take the different amino acids of a protein, I mentioned that there are 20 different kinds, that these 20 kinds of amino acid can be classified in various ways.
27:42
And one way of classifying them is to say that some of these polypeptide chains are non-polar and the others are polar. If we have a polypeptide chain here, like this,
28:05
then we have some side chains, like this one, which is called phenylalanine, or we have ones like this, called valine, in which all the groups are hydrocarbon groups.
28:27
These are the so-called non-polar residues. This means this kind of group of atoms does not like to be in water.
28:40
Molecules of this kind are insoluble in water. They are non-polar, or sometimes called hydrophobic. There are other kinds of side chain in which there is an electrically charged group, which can be negative or it can be positive.
29:06
And groups of this kind are polar ones, hydrophilic. They like dissolving in water. So in a protein, we have a mixture. We have some non-polar groups, some polar ones. These ones do not like water.
29:22
These ones do like water. And the physical chemists suggested many years ago that perhaps in the protein molecule, the non-polar residues are inside and the polar residues are outside, sticking towards the water.
29:41
It is exactly the same kind of situation they suggested, as you have in a soap, where we have molecules which are non-polar at one end and at the end have a polar group, a charged group, positive or negative.
30:02
And in the soap, my cell, one has these groups sitting in such a way that the charged groups are out towards the liquid and the non-polar groups are away in the middle. And the force which holds this object together is essentially a consequence
30:27
of these non-polar groups which do not like water. They try to escape. They try to get away from the water, so they hide themselves inside the complex, leaving the polar groups on the outside.
30:44
It turned out, when we had our model of myoglobin here, we were able, of course, to look at each amino acid along the chain and see where the polar groups and the non-polar groups were.
31:01
And indeed, it turns out, as one would expect on this theory, it turns out that the polar groups are almost all on the outside of the molecule, the non-polar groups are on the inside. So it seems that the physical chemists were correct
31:21
that indeed the force which holds the whole thing together, the forces are similar to the forces holding together a soap, my cell. In other words, it is a case of the non-polar groups escaping from the liquid, the water environment,
31:43
which they do not like, in which they are insoluble. But it is not sufficient only to ask what is the overall force which determines this structure. We have to ask also what are the specific forces
32:02
which determine that it folds up just in this way and not in some other way. And obviously here, when we have our model, we start studying the model and try to understand what it is that causes particular arrangements of the polypeptide chain.
32:24
We notice, for example, that the polypeptide chain begins as a helix or spiral at a certain point. The spiral is broken. The direction of the chain alters. There is a small length of chain which is irregular, non-helical.
32:47
After that, another helix begins. And one might suggest that one should look at the point at which the helix terminates at the break in the helix to see if there is something special
33:01
about the arrangement of amino acids at that point which could account for this particular change in direction or change in the angle of the chain. And it's been disappointing to us to find that it's really difficult
33:22
or impossible by looking at that model to guess what are the forces responsible. The polypeptide chain in myoglobin consists of eight lengths of helix and in between the eight lengths of helix,
33:41
there are seven non-helical regions. Unfortunately, they are all different. If you found that, say, one type of non-helical region was repeated several times in the myoglobin molecule,
34:01
you might say, well, perhaps this is a standard method for turning the corner in a protein and we would expect to find this in other proteins too. Unfortunately, all the seven corners are different and it is extremely difficult from looking at these corners
34:21
to form any impression of the forces responsible. Another way of looking at this would be to say if we had a very good computer, could we put into that computer all the information simply about the sequence of the amino acids along the chain
34:44
and could we predict what its three-dimensional arrangement would be? Well, I think this will be possible in the future, but certainly at the present time it is too difficult to solve it.
35:01
And this brings us back to the problem with which I began, the problem of genetics. We have seen how the genetic apparatus, the DNA, determines the sequence of amino acids in the protein molecule.
35:21
The question is, does the genetic apparatus also determine the three-dimensional arrangement? The geneticists believe that it does not do so, that the genetic apparatus only determines the sequence of amino acids, that the messenger RNA, the ribosomes,
35:44
they make simply a sequence of amino acids and that after this the chain folds itself up. In other words, they suggest that all the information required to determine the three-dimensional structure
36:01
must be contained in that sequence. This is simply a hypothesis. There is no proof of this. Indeed, the hypothesis was invented in the first place simply because it would make life too difficult to understand.
36:20
One could not understand at all how it could be done in any other way. If you wish to determine the three-dimensional structure by some direct mechanism, it would be like mass production in a factory. You would have to have a kind of mold or three-dimensional template on which to fold the molecule.
36:45
And I think you can easily see from the complexity of the structures I've indicated, it's very hard to imagine how such a three-dimensional mold could exist. So the hypothesis that the folding is spontaneous,
37:03
this hypothesis was made simply because any other system seems very hard to believe. And indeed, some evidence has recently come from the biochemists that the information in the sequence certainly is sufficient
37:25
to allow the polypeptide chain to fold itself up. You can take an enzyme which has a specific biological function which catalyzes a particular chemical reaction.
37:42
You can take that enzyme. You can test its activity on the specific reaction it catalyzes and you can then destroy its three-dimensional structure. This is the process known to the biochemists as denaturation of the protein.
38:03
And you can show that after the denaturation, the specific structure has been lost, the activity, the enzyme activity has also been lost. Now in the last few years it has been shown, I think conclusively, that this process certainly can be reversed in the laboratory.
38:26
You can take an unfolded, denatured protein and by suitable experimental arrangements outside the living cell, you can allow that protein to refold itself
38:41
and you then recover the 100%, in one case at least, of its original activity, its original biological activity, and by all the tests the molecule is as good as new. This shows, I think, that indeed the polypeptide chain,
39:05
the sequence of amino acids, must contain enough information to allow the folding to take place spontaneously. Outside the living cell it is not so efficient as it is in the living cell.
39:20
The living cell takes a matter of seconds or the order seconds to manufacture a complete protein molecule. In the laboratory the refolding process takes perhaps 10 or 20 minutes. It is not as efficient as in the living cell. Nevertheless it does work.
39:43
The suggestion, in other words, is that the three-dimensional structure which we study, the biologically active structure of the molecule, is indeed the most stable structure from the thermodynamic point of view.
40:02
I think we can take it that indeed the folding process is spontaneous but we still cannot understand how it takes place because looking at the myoglobin structure
40:21
we find it is too complicated to discover which particular interactions are important in determining the structure. And at this point, of course, we would like to have models in atomic detail of many other proteins available
40:40
so that we could compare them, so that we could deduce some general principles on this about the construction of the molecule. Unfortunately at the present time we still have no other proteins whose structure is known, whose structures are known in atomic detail.
41:03
We cannot carry out this comparison. But one thing we can do is to look at this other molecule I mentioned earlier at hemoglobin. Here is my colleague Max Perutz's model of the hemoglobin molecule.
41:24
Now hemoglobin, as I said, is more complicated than myoglobin. It has four times as many atoms in it, approximately. And so the model which Max Perutz has obtained of hemoglobin is not yet so detailed as that of myoglobin.
41:45
He is looking at the molecule with a rather bad pair of spectacles. Here is the model he has. It is so complicated it is difficult for you to understand
42:00
what is happening here, but hemoglobin is made up of two different kinds of polypeptide chain, the so-called alpha chain and the beta chain. There are two alpha chains, they are white here. There are two beta chains, they are black.
42:22
And then we have the four heme groups. You can see a heme group there and oxygen marked on it. There is another heme group and the other two you cannot see. They are behind. Now, when Max Perutz first obtained this model,
42:42
he, so to speak, took it to pieces in order to see what each individual chain looked like. And here is that model taken apart. You see, here are the two white chains, the two alpha chains.
43:01
Here are the two black chains, and you can put it together and make the whole molecule. And the next thing was to look at the individual alpha and beta chains. Next slide, please. Here are the two chains of hemoglobin,
43:22
the alpha chain and the beta chain. And for comparison, we have put on this picture the chain of myoglobin at the same kind of resolution. And the interesting thing, the very remarkable thing, which Max Perutz discovered at this point,
43:42
was that if you arrange these three chains in the proper manner, then they are exactly the same. You can easily see, we can start here and you go around in this complicated arrangement, that is the alpha chain of hemoglobin, but if we go in myoglobin, we have exactly the same arrangement.
44:04
This is very remarkable. This strange irregular shape is evidently not accidental. Because we find it here in two quite distinct proteins, one protein from the blood, one from muscle cells,
44:23
and furthermore, these proteins came from very different animal species. The hemoglobin was horse hemoglobin, the myoglobin came from the muscle of a whale. So, it looks as if this shape is in some way important.
44:43
It is a common arrangement for different proteins in different animal species. Now, we still do not have a detailed atomic structure for the hemoglobin chains,
45:00
but because these arrangements are so similar, it means we ought to be able to compare the amino acid sequences of the myoglobin chain and of the two kinds of hemoglobin chain. We ought to be able to lay these sequences alongside one another
45:23
and see where the same amino acid appears in all three chains. Because if it is true that certain amino acids are responsible for determining this structure, and since the structure is the same, or almost the same,
45:44
in all three chains, then those amino acids which are important should be identical in all three chains. Now, the strange thing is, when you come to do this, is to find how very few correspondences there are.
46:06
The next slide please. Out of each chain has about 150 amino acids in it. Only 15% of these amino acids are the same,
46:23
are homologous in the myoglobin and in hemoglobin chains. This is a very surprising result. It is surprising that so few of the amino acids correspond and yet the whole structure of the three chains,
46:43
the whole structures are so similar. And of course we have been trying to look at the structure of the molecules and understand why it is that these 15% are important. Certainly you would guess that the important amino acids
47:04
would not be the ones outside the molecule, the ones which simply stick out into the liquid. You could make alterations in those amino acids without upsetting the internal structure. You would expect that it would be some of these internal non-polar residues
47:24
which would determine the way the chains would pack into one another and you would expect that these would be the ones which would be homologous. And indeed it turns out that if you look at the internal amino acid residues,
47:41
the ones inside the molecule, you find that the proportion of those which are homologous is much higher. But even so it is only one in three and I must say that for me this is still quite a mysterious thing. This is part of the problem which we absolutely do not yet understand.
48:06
I do not understand myself how it can be that these chains are so similar when only 33% of the amino acid residues inside the molecules are identical.
48:22
Of course the resemblances between these chains of hemoglobins and myoglobins, these resemblances are interesting not only from the structural point of view but also from the point of view of the systematic zoologist.
48:43
In the past of course the zoologists had to make classifications of animal species on the basis of external characteristics. Now that we begin to see the detailed chemical structure of the important molecules in living cells,
49:03
we can begin to think about a better method of classification, a kind of chemical taxonomy, and we can see whether the relationships between animal species in some way correspond to the relationships between the amino acid sequences of the proteins
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of which they are made up. And indeed it turns out that if you look at the hemoglobins of two very closely related species,
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you find there are very few differences between them. I think it was in Dr. Pauling's laboratory, it was shown a year or two ago, that there are only one or two differences between the amino acid sequence of human hemoglobin and the sequence of gorilla hemoglobin.
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Human beings are very closely related to gorillas. On the other hand, if you compare a human hemoglobin with a hemoglobin from a horse, you find there are something like, I think, 18 differences between them.
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And you can, on this basis, as indeed Dr. Pauling has done, you can construct a kind of molecular evolutionary tree, you can relate the changes which have taken place in the amino acid sequences of the protein,
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you can relate that to the evolutionary tree of the system of animals you are considering. You can suppose, in other words, that there was in the beginning a kind of primitive hemoglobin from which divergence has occurred in parallel with the general evolutionary divergence of the species.
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Well, so far I have only talked about the molecules of hemoglobin and myoglobin as representative protein molecules. We must not forget that these molecules also have a special function, namely to carry oxygen.
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And we might ask, how is this oxygen carried in these molecules? Well, I mentioned that the oxygen is carried on the iron atom of the heme group.
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And I think one can see something of the central problem that we are trying to solve here, for now we are beginning to discuss the function of the protein molecule. The central problem can be exemplified in this case by asking, how is this oxygen carried?
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You see, you can separate the heme group from the protein. You can take the molecule apart, separate the polypeptide chain from the heme group. The heme group alone is a well-known chemical compound,
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and what you find is that if the heme group is separated, it will not perform this trick of combining reversibly with oxygen. But you can push it back into the protein and then its properties are in some way changed
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so that now it does reversibly combine with oxygen. And the question is, how is this done? And this of course is another reason for determining the structure of a molecule like myoglobin because with this structure the theoretical chemists have something concrete
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with which they can try to understand this process of oxygen combination. Indeed, you can perhaps try to construct a model system. Some years ago, before we had this myoglobin structure available,
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some years ago Dr. Wang in the United States decided to try to make an artificial hemoglobin, a kind of ersatz hemoglobin. And what he did was to take no protein at all. He took the heme groups alone.
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He associated those heme groups with these amino acids called histidine. I mentioned earlier that the heme group is attached to the protein in hemoglobin by histidine. So he took heme group and histidine and he embedded this complex of heme group and histidine
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in a matrix of polystyrene, that is to say a synthetic polymer. He chose this because for various theoretical reasons we needn't consider, he thought that the secret might be to put the heme group in a medium of low dielectric constant.
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So he put the heme groups into polystyrene. He found that once they were there they would indeed combine reversibly with oxygen.
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He had in fact constructed a kind of artificial hemoglobin. Here is part of the myoglobin structure. Here is the heme group. And you see it now turns out that Dr. Wang's model was a very good one because in myoglobin and hemoglobin the heme group is associated with one histidine here,
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another histidine there. This corresponds to the Wang model. And furthermore, the heme group is surrounded on all sides by a large number of non-polar residues.
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Here is a phenylalanine, another phenylalanine, and here is I think a valine, and that looks to me like a leucine, and so on. One can go all round. You find it is in a non-polar environment. In other words, speaking electrically, it is in a medium of low dielectric constant.
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So the Wang model was a good one and it worked. But of course now what we must do, or what the theoretical chemist must do, is to take this structure in detail and understand how it is that when this heme group is put into this complicated environment,
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how precisely it does succeed in reversibly combining with oxygen. And indeed the story is a little bit more complicated than I have told you because in fact the way in which hemoglobin and myoglobin combine with oxygen are rather different.
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If you make a curve in which you plot the amount of oxygen taken up by the protein against the pressure of oxygen,
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you find for myoglobin you get a curve of this kind, a hyperbola. This is what you would expect in a molecule containing a single heme group,
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a single iron atom combining with one oxygen molecule. So this is what you get for myoglobin, but for hemoglobin you get a different curve. The curve is now shaped like an S. And from the point of view of the physical chemist, this means that this curve is different from this one
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and it means you notice that at any given pressure of oxygen there is less oxygen on the hemoglobin molecule than there is on the myoglobin molecule curve heme group.
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It means that there is some kind of interaction between the different heme groups on the hemoglobin molecule. It is as if the first oxygen going into the hemoglobin molecule in some way makes it more easy for the other three oxygens to go on later.
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Number one attaching to hemoglobin makes it easier for numbers two, three and four to go on. And we might ask, how is this interaction achieved?
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It is not at all obvious by looking at Max Perutz's model, how it's achieved. You may remember that the heme groups are in little pockets on the outside of the hemoglobin molecule. They are very far apart from one another. And quite recently, Perutz has been able at least to show something of the way this happens,
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because he has compared the structure of hemoglobin with oxygen and the structure of hemoglobin without oxygen. And he finds that they are a little different from one another.
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What happens is that the molecule actually changes size when the oxygen goes on. You might expect perhaps that when the oxygen went there, the molecule would get bigger because we now have the protein plus four oxygen molecules.
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In fact, the reverse takes place. The oxygen goes into hemoglobin, the molecule becomes smaller. It is a kind of breathing of the molecule in reverse. Breathing is when we take oxygen into our lungs and our chest becomes bigger.
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You might expect, it seems perhaps appropriate, that the breathing molecule should breathe like a human being. But it does it in reverse. When the oxygen goes in, the molecule gets smaller. And the way this happens, we have the...
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four chains arranged, Perutz has shown that as the oxygen goes in these chains move relative to one another in a special way so that when the oxygen is there they are a little closer to one another. Clearly the interaction between the four chains which produces this change in the oxygen
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uptake curve, this interaction must in some way be connected with the breathing of the molecule the way in which the chains move relative to one another. And here again is an opportunity for the theoreticians to understand the
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process. Now I have tried to tell you in this lecture something about the structures of these molecules and in particular I have tried to tell you that understanding the structure has not given us the answer to all the questions
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we would like to understand. In fact in some ways knowing what the structure is like has made the questions seem more difficult. We have this problem how does the molecule fold itself up? How is the specific structure determined? We do
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not know. We have this question how are the properties of the heme group modified when they go into the protein molecule modified in such a way that the oxygen combination happens, reversible combination happens at all? We
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do not know except in very general terms. And finally we have this question about the particular arrangement in hemoglobin. How is it that the shape of that curve is determined by the fact that when the oxygen goes into the molecule the molecule changes its shape? So that this field of protein
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structure is by no means a closed one. In fact we are really just at the beginning. I think the situation can be summarized by saying we are now for the first time in a position to be able to make a serious attempt to
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answer some of the questions which are interesting to the geneticists to the biochemists. We are in a position to answer those questions but in fact none of the questions have been answered yet. Thank you.