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Zinc finger binding
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Zinc finger binding
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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 |
When [Carl] Pabo went back to refine this structure, of course he found this. It turned out that he'd actually seen this interesting crystallography, he's a very pure crystallographer and if he doesn't understand the result he doesn't publish it. When he looked at his map, and he told me this, and we could see it in his map anyway, he could see that there were interactions with the second strand but it was from a... it was from a glutamate to a cytosine. Now the glutamate is negatively charged, now it interacts... and so... now we, I knew this but I also knew that in other crystal structures of certain... what do you call them? Restriction endonucleases you've sometimes got this interaction and I don't know how it came about because it seemed to defy the laws of chemistry, but Pabo saw it and he didn't put... he didn't say anything about it, just omitted to mention it. But when you refine the structure to 1.98 resolution he saw very clearly what... well, that was done some years later. It's very cunning, what happens is that the glutamate, I'm sorry to go into such detailed stereochemistry but it's one of the things you really have to understand deeply if you're going to work in this field, the glutamate has a couple of aliphatic residues before you get to the acidic residue. And what Pabo didn't know was that the five six bond of cytosine is hydrophobic. And so, these aliphatics... the binding is not to the... it's not hydrogen bonding at all, which as most of the other interactions are, its hydrophobic bonding and it involves interaction between the aliphatic residues of glutamate and the five six bond of cytosine. And I suspected that because I knew about the five six bond of cytosine from our tRNA works. And then the acidic part bends backwards and binds to the backbone of the protein... so then Pabo published that later. So... but there are many different ways you can recognise a cytosine. Valine will do it because it's a methyl group, so will alanine and so on. So in the end we did work out a stereochemical code but it's very complex so you... so in the end you would have to... I couldn't write down by that time, write down by eye the sequence of zinc fingers which will bind the DNA but it won't be the highest binding. To find the highest binding you've got to make combinations of those different sites, that's really... that's really what it amounted to. That's why the selection... so thought we used to write, particularly Yen Choo, we always talk about tailor-made proteins... they were selected proteins, selected zinc fingers.
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: Refining the structure of zinc fingers
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: Carl Pabo, Yen Choo
Duration: 3 minutes, 10 seconds
Date story recorded: July 2005
Date story went live: 24 January 2008