History and the X-ray Analysis of Protein Crystals
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Lindau Nobel Laureate Meetings135 / 340
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00:00
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Transcript: English(auto-generated)
00:12
Professor Hoppe and friends, I find Professor Hoppe's introduction very useful to me.
00:21
I shall be illustrating some of the remarks he has made in the course of this lecture. As you have heard already from other speakers, discoveries often get lost in the literature.
00:41
Well, perhaps Dick Singh really showed you how to find them however far back you had to go. And sometimes observations that should lead on to great developments get made and somehow not used through gaps of 10, 15, 20 years
01:02
before they are really finally put to good use that everybody works on the problems revealed as in the case of interferon yesterday. And these gaps are in themselves quite interesting
01:23
and they occurred in the course of the story of the X-ray analysis of proteins. Protein crystals were observed in plants and in animal tissues during the course of the 19th century.
01:41
And there are many nice drawings of them made by botanists and others who examined biological tissues. The first one on my first slide was made by Professor Schimper and I think from this part of Germany,
02:04
pictures of crystals observed in plant cells and you can see he was looking at them through microscopes and there is one particularly here. Is there a pointer?
02:23
Which you can see he's viewed through nickels in different directions which shows pleochroism and some of them are protein crystals. I'm meaning this one here where he's obviously got his nickels in different directions.
02:43
And another one on the next slide also taken from more than 100 years ago from Pryor's book on Die Blut Kristaller. A very lovely photograph of hemoglobin crystals.
03:04
I think they are dog hemoglobin showing that he was viewing them through the microscope using nickels, turning them around so that the crystals appeared different colored in different directions. And even at that time it was realized that proteins,
03:23
the molecules, whatever they were in these crystals were large. On the next slide there is a little early analysis made, figured in Pryor's book giving a shot at the molecular weight of hemoglobin.
03:41
It isn't quite right in any direction because the analysis is too high. But it gives, you see, a large figure 13,000. It should be more like 17,000 and then the actual molecule is four times that.
04:00
But it was known that there were large molecules in these crystals in the 19th century and observations were made by both Schimper and Pryor that showed that to get these beautiful pictures of crystals you must keep the crystals
04:22
covered with liquid and that they dried or shrank when removed from their mother liquor. Now the next discovery was the discovery made in Munich by Von Laue, Friedrich and Nipping illustrated by the next slide.
04:41
Oh sorry, this is just another one that shows you a crystal actually growing, a crystal of hemoglobin actually growing in a red blood cell. And you can see that the hemoglobin crystal is occupying almost the whole of one of the blood cells.
05:01
It's this, that particular cell, probably the wall was damaged and so the crystal started to grow whereas in the next one you can see the normal appearance and now the photograph which was taken by Von Laue, Friedrich and Nipping in 1912 in Munich
05:23
by passing x-rays through copper sulfate and it shows that x-rays have wavelengths of the order of magnitude of diffracting units in the crystal and that these units must be the atoms
05:41
arranged in a regular arrangement in three dimensions to produce these effects. Von Laue, Friedrich and Nipping didn't go on to work on this crystal. Its structure wasn't solved for more than 20 years. It seemed quite complicated in those days.
06:03
They moved over to a cubic crystal, zinc sulfide and took very beautiful photographs but the actually first use made of these x-ray diffraction photographs was made by a very young man, W.L. Bragg,
06:20
age 24 in England who showed how to work, how to use the diffraction effects to find the relative positions of the atoms in space in sodium chloride. He was helped by the structure having been suggested
06:40
to him by Barlow who in fact had published a proposed structure in 1885, quite correctly, again a long time before. I illustrate this structure on the next slide. By an actual section in the electron density
07:07
in the crystals of sodium chloride, the electrons scatter the x-rays and because they are grouped into atoms in a regular array in three dimensions,
07:24
the interference is partly destructive and from the spectra, one can form a Fourier series as W.H. Bragg first suggested in 1915.
07:43
The spectra provide the components, the terms of the Fourier series. They, from the scattering, separates the terms. They have to be recombined to give you back the pattern which produced them. And the recombination has to be done,
08:02
is usually done mathematically by calculating the contribution of every term observed to every position in the crystal by a mathematical formula. And for this, you have to know the amplitudes of the waves which you can easily measure
08:20
and also their relative phases which are lost in the process but can sometimes be easily recovered. The calculation, though suggested in 1915, was not in fact made till 1926 by Havickhurst in America
08:41
and Duan, who suggested it, pointed out that the phases were, one, known in sodium chloride from Bragg's work, but two could have been inferred because the heavier atom would dominate the effects. Or alternatively, as Bragg could use to begin with, the differences between sodium chloride
09:02
and potassium chloride, where one ion varied in density, would give a direct method of finding the phase relations. And then the picture can be combined and the electrons plotted, the electron density plotted at any density intervals you liked
09:22
to show the arrangement of the atoms. Now, when the experiments on X-ray diffraction were first made, passing X-rays through crystals, it was natural for different people
09:43
in different parts of the world to repeat the experiments. W.L. Bragg was one. But they were also repeated in Japan. And in Japan, for the first time, in 1913, immediately afterwards, X-rays were put through a protein, silk fibers.
10:05
And I think the next slide should show you a photograph of silk fibroin. Now, this is a photograph in which
10:21
the reflections are very fuzzy. Good photographs were obtained first, actually, in Berlin in 1921-22 by, again, a very young man as he was then, Rudolf Brill, who I hope is still alive and living near Munich,
10:45
as he was a year or two ago. And he took the photographs for his dissertation, helped in the interpretation by Michael Polanyi
11:00
and Hermann Mark, who were slightly older in the same laboratory. Okay, and the interpretation was that in these fibers, there must be long chains of proteins, as indicated by Emil Fisher's experiments,
11:22
and that these chains were not quite regular, that the amino acids might not repeat quite regularly. So this isn't a perfect crystalline photograph, but one in which the essential intervals
11:41
shown by these fuzzy spots are the intervals in the chains. And the next slide shows Professor Mark and Meyer's idea of what the protein chains, amino acid chains,
12:02
should be like to give the actual observed distances between these fuzzy reflections on the silk fibrin photographs. Extended zigzag chains, ultimately glycine and alanine in the fiber structure,
12:25
running through the unit cells. In this period of the 1920s, there were a number of experiments in which crystals were actually prepared in the laboratory
12:42
from newly isolated enzymes and hormones. Urease, Sumner and Northrop pipsin, insulin by J.J. Abel in America. And it was natural for young crystallographers
13:03
in the 1920s to try to put X-rays through these crystals too. So in the laboratory of W.H. Bragg at the Royal Institution, several attempts were made to get X-ray photographs
13:21
of insulin, hemoglobin, one of the enzymes. Edestine, a plant hormone. And they all got nothing but somewhat vague blurs. And two of the young men present
13:41
were Astbury and J.D. Bernal. And when they left the laboratory of the Royal Institution, Bernal for Cambridge and Astbury for Leeds to work on wool fibers at York,
14:03
they were both very anxious to work on proteins. And they corresponded with one another and their correspondence exists in the Cambridge University Library where I found it. And Astbury described how he wrote to Northrop
14:22
for pipsin crystals and Northrop sent him ones. And he got absolutely nothing on the photographs except a sort of, got two rather diffuse reflections rather like some of the silk fibroin ones. And he took fiber photographs as well.
14:43
In fact, that particular silk fibroin photograph is taken by Astbury and not by Brill or anyone of the earlier workers. He found that protein fibers in general tended to give two patterns. One, hair when it was unstretched with two reflections
15:06
which he called the alpha pattern. And then if you pulled it out, it gave the pattern that suggested stretched chains, the beta pattern. But he was very anxious to work on crystals
15:20
and to collaborate with Bernal. The only thing he complained of in his letters was he would like to start a serious collaboration. If only you were not such a soft-hearted chap and taking on problems for all sorts of other people. And the problem of course that J.D. Bernal was taking on at that particular moment
15:43
was the structure of the sterols. He had just put X-ray photographs, X-rays through calciferol cross crystals and shown from his results that the Wieland-Wendaus formula couldn't be correct
16:00
and so opened the way for a whole new passage in sterile chemistry. But Astbury wrote, why not ask for hemoglobin crystals? Adair is the bloke. And Bernal I think hesitated a little
16:21
but suddenly the crystals were brought to him in his hand. They were brought from Uppsala where they had been grown by a young man called John Philpott who was a biochemist learning how to purify proteins with Tizanias.
16:43
And John Philpott enjoyed skiing. He went off skiing in the mountains for a fortnight leaving his crystals growing in the fridge. And when he came back he found his tubes of purified pepsin full of the most marvelous
17:03
large crystals about two millimeters long. And as good fortune for the advance of science would have it, they are passed through the laboratory playing Millikin, the son of R.A., electron Millikin
17:22
who was working in Cambridge on fast reactions and he was shown the crystals and he said, I know a man in Cambridge who would give his eyes for those crystals. And Philpott happened to know the same man, John Desmond Burnell, because he had earlier
17:44
been involved in the isolation of vitamin D at the Medical Research Institute in our country. And so he very willingly handed him a tube of the crystals which Millikin stuck in his coat pocket
18:01
right way up, crystals still in their mother liquor and took them back to Cambridge. And the year was then, 1933. And when Burnell saw the crystals, of course he immediately did, he looked at them first
18:23
within the tube under the microscope and saw they were brightly shining, brightly birefringent and he took one out of it being in a hurry to see what was happening just with a needle out of the tube and took an x-ray photograph of it
18:44
and got exactly what Asprey had got, only perhaps rather less because he was a less skillful in general experimenter, hardly anything on the photograph and he thought, this must be wrong.
19:00
Went back and looked at the crystals, brighten their mother liquor and it suddenly struck him that they needed their mother liquor round them to keep their actual form. And he was lucky in another way because he was working at that time also on the problem of ice and water
19:24
and he had in the laboratory a student, Helen McGore, taking x-ray photographs of ice crystals which she grew in little fine Lindemann glass tubes and kept at low temperatures.
19:42
So Burnell took just one of her little fine walled tubes about half a millimeter across and fished out a pepsin crystal within its mother liquor, concealed the tube at two ends and put x-rays through it and immediately got an x-ray photograph
20:02
with reflections, ever so many reflections all over the photograph. Now the next slide should, if I'm remembering right, well first we have the people. So here is J.D. Burnell much later on in life
20:22
and talking to Katie Dornberger, a student who was working at that time with V.M. Goldschmidt in Goettingen but came to work with him and came to work on some of the protein problems later and myself, and I must say this photograph
20:41
was taken in relatively old age when Kate had just become the director of a small institute for x-ray diffraction studies. In Berlin East.
21:01
Now the next slide shows another character in the story whom I will mention, A.L. Patterson in his laboratory with two students and the next slide shows the photograph.
21:22
This isn't the original pepsin photograph. Here are some pepsin crystals soothing about in their mother liquor and above is a photograph taken of them by Professor Tom Blundell in Birkbeck College London
21:42
showing very, very many reflections along these parallel lines on an x-ray photograph. The original photographs we think must have perished at Birkbeck since the laboratory, part of the laboratory was destroyed
22:01
during bombing during the war but this at least illustrates the character of the picture quite different from silk fibro in a definite crystal repeat. You can very easily measure one lattice constant, 67 angstroms from the separation of the lines
22:21
and the other one, in fact we got it wrong when first we measured it. We got it about half the size it really is. It's really nearly 300 angstroms corresponding to the long dimension of the pepsin crystals. I was at that time working with Bernal
22:48
and it was only a sort of bit of bad luck but perhaps it was good luck for science that I was not in the laboratory the day the first crystals came in.
23:01
I was having a bad cold or something and Bernal made all of the first observations himself. I'm always a little afraid I might have got more on the first photograph since it was possible to get more reflections from the dried crystals than Bernal actually did and so delayed the observation
23:23
that it was absolutely necessary to keep these crystals in their mother liquor. But I went on to take most of the rest of the photographs but I do some of the calculations which we didn't carry very far because our first measurements indicated
23:44
that we had a very large unit cell that it could correspond to there being 12 pepsin molecules in this cell each weight about 40,000,
24:00
several thousand atoms each you see within each molecule and that this was, it was beyond our possible means at that time to think that we could work out the structures of such molecules. And yet the reflections extended
24:26
to about one and a half angstroms. It was clear that they were sufficient to show us atoms if ever we could form an electron density pattern from them and look at it.
24:44
At the time I was under pressure to return to Oxford to a college teaching appointment that should lead to a permanent appointment. I was most unwilling to go
25:01
but everyone in Cambridge said difficult to get university jobs in this time. Of course you must take it. So reluctantly I went back to Oxford. Bernal had got a small grant to support me of 200 a year which he gave to another young person
25:28
and I think, could I have the next slide? And this was Isidore Van Cooken who then was visiting from America who had come over with W.L. Bragg
25:42
and was working with W.L. Bragg at Manchester and heard about the work at Cambridge and wanted to join in and he came in to take the next protein crystal, chymotrypsin from Northrop and then passed over to work on virus crystals
26:04
but played a very important part in the development of the subject and particularly later in America. And the next year there came another young person to work with Bernal in Cambridge and I think he's shown on the next slide.
26:21
And again, much older than he was, Max Perutz and Max Perutz came from Vienna wanting to work with Hopkins but Mark had forgotten to ask Hopkins
26:42
to have Max Perutz as a research student when he visited Cambridge in 1935 because he was so excited by the work that Bernal was doing and sent him instead to work with Bernal
27:01
saying there's someone who really needs you and Max said, but I don't know any crystallography and Mark said, you will learn my boy which he did the hard way for many years to come.
27:23
The other one in the picture is John Kendrew who Professor Hoppe mentioned and he doesn't come into the story for very much longer. Now what happened to me in Oxford going back to begin work all on my own
27:41
was that Sir Robert Robinson who was then professor of organic chemistry was given a small present of the first insulin crystals obtained by the firm Boots in our country
28:01
following a prescription for growing insulin crystals given by D.A. Scott in America that it was necessary to add zinc to the preparation and they gave Robinson 10 milligrams of insulin
28:22
in a little tube and he hadn't any use for them and knew the work that we had done in Cambridge taking X-ray photographs of pepsin crystals so he said, why don't you try to photograph these and they were microcrystalline but very bright and birefringent
28:42
and so I looked up all the preparations and grew the crystals finally not very well by Scott's method large enough to take X-ray photographs of and I made a horrible mistake. I decided that it didn't matter
29:01
whether they were wet or dry and it was easier to handle them dry. I dried them like good organic chemists pouring methyl alcohol over them and then took X-ray photographs of them and these very dry looking crystals as you can see are the crystals, not looking very good single crystals
29:23
but they are and up at the top is the little X-ray photograph they gave. Well they gave an X-ray photograph, spots on the film and I developed the first X-ray photograph about 10 o'clock at night
29:41
and waited in the lab while I fixed it and washed it and then walked out absolutely dazed, very excited little spots on this photograph down through the center of Oxford away from my lodgings and about midnight I was accosted by a policeman
30:03
who said where are you going? So I said not very truthfully back to college and turned around and went back but I woke up in the morning, next morning about six and I was suddenly extremely worried and I went to the, I thought perhaps those spots,
30:24
perhaps those crystals aren't really protein crystals at all but something else, some impurity, some breakdown product in the preparation and I went round very quickly before breakfast to the laboratory and picked one out of the tube and tried protein tests on it
30:42
and I tried the xanthoprotic reaction which consists in dropping first a drop of concentrated nitric acid and it turns yellow and then a drop of ammonia and it turns brown which it did to my great relief
31:01
and I went back happily to breakfast. Now I perhaps should tell all those who are young here why I knew that reaction so well was because when I was rather young and still at school I had a laboratory and did experiments on my own
31:22
and I was doing experiments suggested by Parsons Fundamentals of Biochemistry and completing something or other one Sunday in a nice new silk frog and of course one shouldn't ever do this kind of thing
31:41
and I accidentally dropped a spot of nitric acid on this, the front of my dress and seized the nearest alkali which was ammonia and put it on it where of course it was much worse and I was incredibly upset.
32:02
My mother comforted me and said she could cover it all with a frill which she did and so this particular reaction is indelibly engraved in my mind and I was very pleased when I could test it once again with the insulin crystals.
32:20
Now what happened after that? I didn't really remember until I was back reading the Bernal files at Cambridge but it's obvious that directly after breakfast I rang up the Cambridge lab to tell them that I had taken these insulin photographs and got the very sad news that Bernal was at home
32:43
with a temperature of 104. So then I wrote a little letter to his wife saying please tell him when he's well enough that I have these insulin photographs and I gave the rough dimensions of the crystal
33:02
unit cell and there on the next slide a ROM is the real form of the crystals, 74.8 across, 30.9 high and within the crystal there's roughly 36,000
33:24
molecular weight of protein and it should formally be divided into three which by the crystal symmetry to give you 12,000 molecular weight. For the insulin molecules in the unit cell.
33:46
Bernal recovered and wrote me a letter which begins, Dear Dorothy, Zn naught point five two percent.
34:03
Co naught point four nine percent. Cd naught point seven four percent. This gives rather less than three in each case.
34:20
I am going back to Cambridge on I forget when. I will send you some cadmium stuff. And any crystallographer can see what he was saying in this letter. This zinc according to DA Scott's observations
34:40
is replaceable by other elements of which cadmium is the heaviest so far observed. You should try and see if the cadmium crystals show changes in the intensities of the x-ray reflections and then you might be able to use
35:02
the method of isomorphous replacement to determine phase constants for the different reflections and really see the atoms in your crystal. Terribly premature I'm afraid. Again I didn't remember all the details.
35:20
I found the letter in which I said, sorry I can't get anything like good crystals from the cadmium material. It must be very impure. I'm having a terrible time with scholarship examining for the college.
35:41
And I did make one or two abortive efforts but I had a feeling that cadmium wasn't really heavy enough to do what was wanted. And that anyway if I did a little calculation on the number of atoms that there were in insulin it was too large a problem for myself to set out
36:04
to work on at the age of 24. And that I must try to solve some simpler structure first. I tried out the idea of the isomorphous replacement game
36:23
actually in little calculations at the royal institution in a notebook on cholesterol, chloride and bromide. While I was taking the insulin photograph which is shown on the previous slide
36:41
because it's a very, I took that particular photograph for show for publication in the royal society using the very big x-ray tube at the royal institution for the purpose. So I didn't go on with insulin
37:00
but Naxp roots went on with hemoglobin. Could I have the next slide please? I went on as I said with sterols and with the sterols I explored the possible use of both heavy atoms and isomorphous replacement for showing electron densities.
37:22
And this is one of the experiments we did and I show it because I have to introduce another character in the story and this is A.L. Patterson. A.L. Patterson I showed on that earlier slide was one of the young men, A.L. Patterson,
37:43
Asprey and Bonnell who are all.