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Views | Duration | ||
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61. Solving the structure of a two-zinc finger construct | 77 | 06:53 | |
62. Repertoire selection technology | 70 | 04:21 | |
63. Phage display and solving the 'mystery' of the stereochemical code | 63 | 05:57 | |
64. Refining the structure of zinc fingers | 58 | 03:10 | |
65. Zinc finger binding | 95 | 01:03 | |
66. Intervening in gene expression for the first time | 70 | 07:14 | |
67. Trying to improve the zinc finger constructs | 74 | 03:09 | |
68. Experimenting with zinc finger constructs | 69 | 03:15 | |
69. Yen Choo's company: Gendaq | 495 | 03:25 | |
70. Making zinc finger archives | 88 | 02:52 |
The first thing we did was to, I had by this time introduced two dimensional; NMR into the lab, I'd recruited, against the opposition of some of my cryptographic colleagues, NMR structure determination, got a man called David Neuhaus to come here. In the lab... I wanted to do this for many years while Sydney [Brenner] was director but he couldn't bring himself to do it, he didn't really... himself feel the need for it. Also, the crystallographers object to that, they used to say... well, can you solve a structure more than 20,000 daltons? And of course, you couldn't at that time. They said, 'Well, that's not of interest to us.' But once I was director I found some allies, Tom Creighton for example wanted to know the structure, different folding forms, intermediate forms of BPTI used in the folding of a protein, so I had a bit of support in the lab I couldn't one sidedly just fix the... the lab is... well I was director but acted more like a chairman. So I did recruit structural studies so we had David Neuhaus in the laboratory and so we decided to solve a structure of a zinc finger.
Now, at that point I made an unwise decision, I said, 'Look, we're not going to need to know the structure of a zinc finger, we need to know how the zinc fingers are joined together by this linker.' It seems to be that it would be highly flexible and we didn't know that, so I suggested he solve the structure of a two-zinc finger construct which we made, two-zinc fingers is Y5. Unknown to me at the Scripps Institute Peter Wright had... was working on the structure of the single-zinc finger, so they published first and the NMR structure of a zinc finger showed very clearly what we had proposed, that the zinc was holding the two parts of the protein together and the two cysteines came off a beta sheet of two turns, two beta strands and the two histidines came off a helix, three amino acids apart, we could predict that, I could see that something like that was about to happen. And David Neuhaus republished later showed that the two-zinc fingers were totally independent solution, you could solve them as two separate fingers and you could work out their different orientations from the NMR and that covered the whole range, so what he proved was the two fingers had identical structures, to within less than a fraction of an angstrom, though the sequences were somewhat different. Of course, they have seven conserved residues and so it was I had basically predicted, zinc bound to the two cysteines, the two histidines and the three hydrophobic acids formed the kind of core, structural core, so... and Neuhaus showed that the linker was flexible so therefore it could end position itself on any sequence of DNA.
And then we also showed by... we'd also... before that I've got to say we'd already showed by EXAFS that the environment of the zinc was two cysteines and two histidines, we knew that from EXAFS, that happened in Manchester. We did this in Daresbury, by EXAFS, but then we still didn't have a crystal structure of the zinc finger with DNA and by this time a lot of people had joined in this field. Jeremy Burke had proposed a structure based upon the idea of three conserved hydrophobic groups and cysteines and histidines and based upon the structure, pherodoxin, Jeremy was at Johns Hopkins, proposed a structure which turned out to be basically correct in outline and the NMR structure showed that he was correct, so it's not often that you get a theoretical direction but he used the three conserved hydrophobics and showed that they packed... that preceded... the NMR work. I must say, by this time, a lot of people had joined in and Carl Pabo who was then the MIT, and there was one that Chris Lock was working on, transcription factors together with a young man called Nikolai Pavletich, a student who has then gone on to great heights, solved the structure of the... complex of the three-zinc finger peptide from a gene called ZIF268 which was a DNA binding domain. The DNA binding domain was ZIF268 which was an early response gene and showed very clearly that the three fingers bound in the major group of the DNA, they wound and wound the major group and that each finger bound to three bases and... three successive bases so that the three fingers bound to three base pairs. And so it's one finger recognises and binds to three base pairs. These were all on one strand of the DNA and that the fold, of course, the fold was a tremendous mark that the fold was NMR work, that was correct. Now, the three sites on the helix is the recognition helix, and positions minus one, position three and position six on the helix, are where the amino acids are, so you then can of course generate diversity by putting different amino acids onto the three positions. But the structures showed that it couldn't be alphabetically, because there isn't enough, and so we... so for example, the structure showed that although one... if we had a guanine it could be recognised, a guanine in the DNA sequence it could be recognised by either arginine or by a lysine. That was if you had them in positions one or three of the triplet, but if you had position two in the triplet then you had to use a histidine but there's not enough room for a long residue to get in.
Born in Lithuania, Aaron Klug (1926-2018) was a British chemist and biophysicist. He was awarded the Nobel Prize in Chemistry in 1982 for developments in electron microscopy and his work on complexes of nucleic acids and proteins. He studied crystallography at the University of Cape Town before moving to England, completing his doctorate in 1953 at Trinity College, Cambridge. In 1981, he was awarded the Louisa Gross Horwitz Prize from Columbia University. His long and influential career led to a knighthood in 1988. He was also elected President of the Royal Society, and served there from 1995-2000.
Title: Solving the structure of a two-zinc finger construct
Listeners: John Finch Ken Holmes
John Finch is a retired member of staff of the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK. He began research as a PhD student of Rosalind Franklin's at Birkbeck College, London in 1955 studying the structure of small viruses by x-ray diffraction. He came to Cambridge as part of Aaron Klug's team in 1962 and has continued with the structural study of viruses and other nucleoproteins such as chromatin, using both x-rays and electron microscopy.
Kenneth Holmes was born in London in 1934 and attended schools in Chiswick. He obtained his BA at St Johns College, Cambridge. He obtained his PhD at Birkbeck College, London working on the structure of tobacco mosaic virus with Rosalind Franklin and Aaron Klug. After a post-doc at Childrens' Hospital, Boston, where he started to work on muscle structure, he joined to the newly opened Laboratory of Molecular Biology in Cambridge where he stayed for six years. He worked with Aaron Klug on virus structure and with Hugh Huxley on muscle. He then moved to Heidelberg to open the Department of Biophysics at the Max Planck Institute for Medical Research where he remained as director until his retirement. During this time he completed the structure of tobacco mosaic virus and solved the structures of a number of protein molecules including the structure of the muscle protein actin and the actin filament. Recently he has worked on the molecular mechanism of muscle contraction. He also initiated the use of synchrotron radiation as a source for X-ray diffraction and founded the EMBL outstation at DESY Hamburg. He was elected to the Royal Society in 1981 and is a member of a number of scientific academies.
Tags: David Neuhaus, Sydney Brenner, Tom Creighton, Peter Wright, Jeremy Burke, Carl Pabo, Chris Lock, Nikolai Pavletich
Duration: 6 minutes, 54 seconds
Date story recorded: July 2005
Date story went live: 24 January 2008