John Kendrew (1964) - Recent Studies of the Structure of Proteins

Ladies and gentlemen I feel very happy that even if I was late for all the other lectures, at least I arrived in time for my own. And I am happy also to be here in Lindau, I feel like what we call in Cambridge, a freshman, a first year student of Lindau 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 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. Or if one is writing in German one should say DNS, desoxyribonukleinsäure and that this molecule in the chromosome of the living cell and the nucleus carries the hereditary information which comes out from the nucleus in the form of another related molecule which is called messenger RNA or ribonucleic acid. And this molecule attaches itself to the so called ribosomes. And there it makes protein. 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 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. 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. 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. 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 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. 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. The lights please. We have the long protein chain. The polypeptide chain, 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. 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. 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 of converting some substance A into another substance B. This is a specific catalytic reaction of the enzymes. And what we shall be interested in today is what is the relation between this polypeptide chain 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 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, 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, especially 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. 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. 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 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. 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, 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 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. 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? 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 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 2 proteins with which I have been particularly associated, the protein haemoglobin and myoglobin. Now just to say a little bit about, to tell you a little bit about these molecules. Haemoglobin 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. It’s an oxygen carrier. And it is a fairly middle sized protein, it has got something like 10,000 atoms in it and those 10,000 atoms are composed of 4 polypeptide chains. And each chain carries a haem group. Now we can look in the next slide at a picture of the haem group. This is a flat group of atoms with an iron atom in the centre and it is this iron atom which carries an oxygen molecule. And haemoglobin has 4 haem groups so it contains 4 iron atoms, carries 4 oxygen molecules. That is one of the 2 proteins that I shall be concerned with and this is the protein which my colleague Max Perutz has been studying for many years. 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 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 haemoglobin 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. 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 but less complicated than haemoglobin. And whereas haemoglobin has 4 polypeptide chains, myoglobin has one chain. It has one haem group and so it can carry one oxygen molecule. 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. 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, 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 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. 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. 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. 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, 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 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. 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, it is in 3 dimensions. It is rather difficult to represent this because our screen is only 2 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 and we pile them on top of one another and so we get a 3 dimensional representation. And here the spectacles with which we look at the molecule are not very good ones. 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, 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. The next slide please. Here is a model of the myoglobin molecule. Again looked at not sharply, we do not see the individual atoms but we could see the way in which this polypeptide chain goes around the molecule. And here is the iron atom and the haem group. You will see it is a very irregular, a very complicated object 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 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, we cannot tell how the atoms in the chain are arranged. The next slide. Here we have the next map of the myoglobin molecule and we were very happy when we saw this map, 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. And this made us realise that as in so many things our colleague Doctor 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. 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. 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. The next. 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. Those of you who are biochemists will perhaps recognise here we have the haem group and the iron atom. The haem group edge on and it is attached to the rest of the molecule by a group here which you can see has got 5 atoms in a ring. And the biochemists will immediately recognise this as the amino acid which is called histidine. And of course many years before we saw this histidine in the model, 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. There are 2,500 atoms, the model is rather complicated. Here is a model of the myoglobin molecule. 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. 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. 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. 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. 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 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 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 haem group which is attached to the rest of the protein by means of this histidine residue here. 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. 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 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 between one piece of chain and another piece of chain. This is not the case in myoglobin, 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? 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 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. 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 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. These are the so called non-polar residues. This means this kind of group of atoms does not like to be in water. 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. 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, 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. 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 have a polar group, a charge group, positive or negative and in the soap micelle, 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 of these non-polar groups which do not like water, they try to escape, they try to get away, find the water, so they hide themselves inside the complex leaving the polar groups on the outside. 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. 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. That indeed the force which holds the whole thing together, the forces are similar to the forces holding together a soap micelle. In other words it is a case of the non-polar groups escaping from the liquid, the water environment 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 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. 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. 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 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 or impossible by looking at that model to guess what are the forces responsible. The polypeptide chain in myoglobin consists of 8 lengths of helix and in-between the 8 lengths of helix there are 7 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 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 7 corners are different and it is extremely difficult from looking at these corners 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. 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. 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. 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, 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 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, 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 mould or three-dimensional template on which to fold the molecule. 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 mould could exist. So the hypothesis that the folding is spontaneous, 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 to allow the polypeptide chain to fold itself up. You can take an enzyme which has a specific biological function which catalyses a particular chemical reaction. You can take that enzyme, you can test its activity on the specific reaction it catalyses. And you can then destroy its three-dimensional structure. This is the process known to the biochemists as denaturation of the protein. 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. You can take an unfolded, denatured protein and by suitable experimental arrangements outside the living cell you can allow that protein to refold itself. 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, 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. The living cell takes a matter of seconds or the order of 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. The suggestion in other words is that the three-dimensional structure which we study, the active, biologically active structure of the molecule is indeed the most stable structure from the thermodynamic point of view. 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 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 so that we could compare them, so that 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 structures are known in atomic detail. We cannot carry out this comparison. But one thing we can do is to look at this other molecule I mentioned earlier at haemoglobin, here is my colleague Max Perutz’s model of the haemoglobin molecule. Now haemoglobin as I said is more complicated than myoglobin, it has 4 times as many atoms in it approximately. And so the model which Max Perutz has obtained of haemoglobin is not yet so detailed as that of myoglobin, 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 what is happening here but haemoglobin is made up of 2 different kinds of polypeptide chain. The so called Alpha chain and the Beta chain, there are 2 Alpha chains, they are white here. There are 2 Beta chains, they are black and then we have the 4 haem groups. You can see a haem group there and oxygen marked on it. There is another haem group and the other 2 you cannot see, they are behind. Now when Max Perutz first obtained this model, 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 2 white chains, the 2 Alpha chains, here are the 2 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. The next slide please. Here are the 2 chains of haemoglobin, the Alpha chain and the Beta chain and for comparison we have put on this picture, the chain of myoglobin at the same kind or resolution. And the interesting thing, the very remarkable thing which Max Perutz discovered at this point was that if you arrange these 3 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 haemoglobin. But if we go in myoglobin we have exactly the same arrangement. This is very remarkable, this strange irregular shape is evidently not accidental because we find it here in 2 quite distinct proteins, one protein from the blood, one from muscle cells and furthermore these proteins came from very different animal species. The haemoglobin was horse haemoglobin, the myoglobin came from the muscle of a whale. So it looks as if the shape is in some way important, it is a common arrangement for different proteins in different animal species. Now we still do not have a detailed atomic structure for the haemoglobin chains. 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 2 kinds of haemoglobin chain. We ought to be able to lay these sequences along side one another and see where the same amino acid appears in all 3 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 in all 3 chains, then those amino acids which are important should be identical in all 3 chains. Now the strange thing is when you come to do this is to find how very few correspondences there are. The next slide please. Each chain has about 150 amino acids in it. Only 15% of these amino acids are the same or homologous in the myoglobin and in haemoglobin 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 3 chains, 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 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 which would determine the way the chains would pack in to one other. 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, the ones inside the molecule, you find that the proportion of those which are homologous is much higher. But even so it is only 1 in 3. 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. 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. Of course the resemblances between these chains of haemoglobins 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. In the past of course the zoologist 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 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 of which they are made up. And indeed it turns out that if you look at the haemoglobins of 2 very closely related species you’ll find there are very few differences between them. I think it was in Dr. Pauling’s laboratory, it was shown a year or 2 ago that there are only 1 or 2 differences between the amino acid sequence of human haemoglobin and the sequence of gorilla haemoglobin. Human beings are very closely related to gorillas. On the other hand if you compare a human haemoglobin with the haemoglobin from a horse you find there are something like, I think 18 differences between them. 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, 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 haemoglobin from which divergence has occurred in parallel with the general evolutionary divergence of the species. Well so far I have only talked about the molecules of haemoglobin and myoglobin as representative protein molecules. We must not forget that these molecules also have a special function, namely to carry oxygen. 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 haem group. 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 is how this can be exemplified in this case by asking how is this oxygen carried. You see you can separate the haem group from the protein. You can take the molecule apart, separate the polypeptide chain from the haem group. The haem group alone is a well known chemical compound. And what you find is that if the haem 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 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 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, some years ago Dr. Wang in the United States decided to try to make an artificial haemoglobin, a kind of “Ersatz”- haemoglobin. And what he did was to take no protein at all, he took the haem groups alone, he associated those haem groups with these amino acids called histidine. I mentioned earlier that the haem group is attached to the protein in haemoglobin by histadeine. So he took haem group and histidine and he embedded this complex of haem group and histidine 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 haem group in a medium of low dielectric constant. So he put the haem groups into polystyrene. He found that once they were there they would indeed combine reversibly with oxygen. He had in fact constructed a kind of artificial haemoglobin. The next slide please. Here is part of the myoglobin structure, here is the haem group and you see it now turns out that Dr. Wang’s model was a very good one. Because in myoglobin and haemoglobin, the haem group is associated with one histidine here. Another histidine there, this corresponds to the Wang model and furthermore the haem group is surrounded on all sides by a large number of non-polar residues. 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 around. You find it is in a non-polar environment, in other words speaking electrically it is in a medium of low dielectric constant. So the Wang model was a good one and it worked. But of course now what we must do or what the theoretical chemists must do is to take this structure in detail and understand how it is that when this haem group is put into this complicated environment, 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 haemoglobin and myoglobin combine with oxygen are rather different. If you make a curve in which you plot the amount of oxygen taken up by the protein against the pressure of oxygen, 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 haem group, a single iron atom combining with one oxygen molecule. So this is what you get for myoglobin but for haemoglobin you get a different curve, the curve is now shaped like an S. And from the point of view of the physical chemists this means that this curve is different from this one. And it means you notice that at any given pressure of oxygen there is less oxygen on the haemoglobin molecule than there is on the myoglobin molecule per haem group. It means that there is some kind of interaction between the different haem groups on the haemoglobin molecule. It is as if the first oxygen going into the haemoglobin molecule in some way makes it more easy for the other 3 oxygens to go on later. Number 1 attaching to haemoglobin makes it easier for numbers 2, 3 and 4 to go on. And we might ask how is this interaction achieved, it is not at all obvious by looking at Max Perutz’s model how it’s achieved. You may remember that the haem groups are in little pockets on the outside of the haemoglobin 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. Because he has compared the structure of haemoglobin with oxygen and the structure of haemoglobin without oxygen. And he finds that they are a little different from one another. 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 4 oxygen molecules. In fact the reverse takes place, the oxygen goes into haemoglobin, 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, 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 these 4 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. And clearly the in interaction between the 4 chains which produces this change in the oxygen 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 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 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 now know. We have this question for how are the properties of the haem 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 do not know except in very general terms. And finally we have this question about the particular arrangement in haemoglobin. 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 structure is by no means a closed one, in fact we are really just at the beginning. I think the situation can be summarised by saying we are now for the first time in a position to be able to make a serious attempt to 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.

John Kendrew (1964)

Recent Studies of the Structure of Proteins

John Kendrew (1964)

Recent Studies of the Structure of Proteins

Comment

John Kendrew was the first scientist who succeeded in elucidating the structure of a protein in atomic resolution. By means of X-ray crystallography he analyzed the structure of myoglobin, the molecule that stores and releases oxygen in the muscle. It contains about 2600 atoms. When he started his work, the largest molecules that had been solved by X-ray crystallography contained but a few dozen atoms. Kendrew had to examine 110 crystals and measure the intensities of about 250,000 X-ray reflections.

In 1946, Kendrew had joined the group of Max Perutz at the Cavendish laboratory in Cambridge. Perutz, the pioneer of protein crystallography, had set out to solve the structure of hemoglobin, the oxygen carrier of the blood, in 1937. Hemoglobin is four times as large as myoglobin, however, and Kendrew reached the finish line one year earlier than Perutz, in 1958. From the beginning, the two scientists worked together very well, as Max Perutz pointed out in a letter to his parents-in-law: „He knew no X-ray crystallography when he came last January, learnt the elements of the subject in two weeks, set to work on his problem which required great experimental skill, and solved it in a few months’ concentrated work. Besides, he is a charming fellow, and it is a pleasure to talk to him; so I consider myself very lucky to have him with me.“[1]

Lindau also could count itself lucky to welcome Kendrew already two years after he had shared the Nobel Prize in Chemistry with Perutz. In a very clear and concise way Kendrew presented a state-of-the-art overview on structural biology as few others would have been able to give it at this time. He introduced the recent findings on myoglobin and hemoglobin, of course, but he mainly directed the attention of his audience to the relationship between the structure and the function of proteins, and intensively dealt with questions like: How does a protein fold up? How is its specific structure determined? Why are myoglobin and hemoglobin quite similar in their structure while only a few amino acids in their sequence correspond? How does hemoglobin change its conformation when „breathing“?

„Understanding the structure has not given us the answer to all the questions we would like to understand“, Kendrew concluded. „This field of protein structure is by no means a closed one. In fact, we are really just at the beginning.“ As a researcher, a teacher and a science manager he continued to bring this new field to fruition. Together with Perutz and others, he founded the European Molecular Biology Organization, and from 1974 to 1982 he served as the first director of the European Molecular Biology Laboratory in Heidelberg.

Joachim Pietzsch

[1] Georgina Ferry: Max Perutz and the secret of life, London 2007, p. 126

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