Диссертация (1173136), страница 58

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за открытие и развитиеиспользования зеленого флуоресцентного белка вместе в Роджером Тсиеном и МартиномЧалфи.Roger Tsien — Роджер Тсиен, лауреат Нобелевской премии по химии 2008 г.; американскийхимик китайского происхождения, профессор кафедры химии и биохимии КалифорнийскогоУниверситета в Сан-Диего.Martin Chalfie — Мартин Чалфи, американский нейробиолог, лауреат Нобелевской премиипо химии 2008 г, член Национальной академии наук США.NobelCeremonie—церемонияврученияоднойизсамыхпрестижныхмеждународных наград — Нобелевской премии, которая проходит ежегодно 10 декабря.Douglas Prasher — Дуглас Прашер, американский биохимик, который впервые выделил ген,кодирующий GFP. В 1985 году он клонировал ген экворина.Frederick Ichiro Tsuji — американский биохимик, чьи работы посвящены свечениюживотных и растений.Ex.

11. Make sure that you know these abbreviations:GFP, green fluorescent protein — зеленый флуоресцентный белок (ЗФБ), выделенный измедузы Aequorea VictoriaUV lamp, ultraviolet lamp — УФ-лампа, ультрафиолетовая лампаEx. 12. A. Read the title of the video lecture “Microscopy: Fluorescent Proteins” and watchepisode I. What do you think this video might be about?B. Which of the following information do you expect to find in the video lecture “Microscopy:Fluorescent Proteins”?a) the discovery of green fluorescent proteins;b) the investigation of properties of the green fluorescent protein;c) the application of fluorescent proteins.C. Watch the video lecture “Microscopy: Fluorescent Proteins” and say whether you haveguessed correctly.Microscopy: Fluorescent Proteins255Roger TsienEpisode I Hello, I’m Roger Tsien, and I am here today to tell you about fluorescent proteins, whichhave made a major impact on microscopy, because they are genetically codable and provide arelatively direct link from molecular and cell biology into colors that we can directly see, particularlyin the light microscope but also, as you will see later on, in more macroscopic levels as well asPrasher D.

[Электронный ресурс]. – Режим доступа: https://www.conncoll.edu/ccacad/zimmer/GFPww/prasher.html255 Tsien R. Microscopy: Fluorescent Proteins (Roger Tsien) [Электронный ресурс]. – Режим доступа:https://www.youtube.com/watch?v=qK9aYnkIr3w. (последняя дата обращения 10.09.2017 г.)254287eventually now beginning to help us with super-resolution.Episode II. So, here this picture is, of course, of the jellyfish, and this jellyfish is where it all began.And in a way, this is the creature that we most have to thank. This is the jellyfish Aequorea Victoria,which was studied in Puget Sound by Professor Shimamura <…>.

This is the source of two of themost actually valuable proteins in cell biology. First, there is the protein that actually enables thisjellyfish to glow, and that’s called aequorin; and when the jellyfish is alarmed in the water, and whenthe water is disturbed, it emits a glow. And then the partner of aequorin is the green fluorescentprotein, which changes the color from blue to green.Episode III.

To this day, we really don’t have a well-accepted explanation as to why the jellyfishwants to glow, why should it show this remarkable phenomenon, when the water is disturbed; nor dowe know why, when the jellyfish glows, why was it so important that it glows green instead of blue.Why not just leave the aequorin, or if it really was important to glow green, why not just changeaequorin in the first place to make it glow green directly rather than invent an additional protein; butwe are very grateful to the jellyfish for having invented GFP.Episode IV.

So, the example – this is perhaps one of the only videos that we know of, where we canshow you the glow of he jellyfish. And this is a jellyfish upside-down in a beaker in an aquarium atAlaska, where the jellyfish can still be found. And (there is actually,) we are illuminating withultraviolet light right now, and you may be able to see there is a circle here of that is got little dotsaround the edge – that is the jellyfish glowing. And this is the green fluorescent protein naturallymade by the jellyfish that we’re exciting with the UV lamp.

I’m sorry the beaker is a bit fluorescent,we should have got a better beaker that didn’t have any, but there’s some yellow background. Butnevertheless, you can see the jellyfish there, and it’s upside-down and confined to this little beaker.Episode V. And what we’re going to do in a moment is to poke it to stimulate the flash. So we’regoing to start here, and you may be able to see that the jellyfish is slightly moving around.

In thisvideo, we are playing with the illumination trying to get it nicely aimed at the jellyfish; and in amoment, we’re going to bring in this rod, with which we’re going to poke it. Then we turn out thelights, and we stir. And during that stirring, that was the light emitted by the jellyfish – not the lightthat previously had been applied from our UV lamp.

So, that’s it. That is the flash. And maybe, youmight have been able to see that it was sort of greenish, maybe.Episode VI. Anyway, the man who discovered that phenomenon is shown here. This is OsamuShimomura, and in 1962, he published his first paper on aequorin. And that was really the protein hewas most interested in. It had the really exciting job of turning chemical energy into light. And healso mentioned that he found this contaminant that got in the way a little bit of the purification, andthat it was a greenish protein that fluoresced green.

And that later he mentioned that it changed thecolor of the jellyfish from the blue of aequorin to the green of GFP. Later in 1979, he actuallyproposed the structure of the chromophore of GFP, based on surprisingly little evidence, but he got italmost absolutely, completely correct. There were only very small modifications that later work thatcame in. This is a picture of him much later in 2008 in the rehearsal for Nobel Ceremony.

And he isactually holding a UV lamp: that’s the violet that is on your left, and then next to it is the tube ofGFP. And this is perhaps the last tube in existence that is actually made from real jellyfish he hadkept in his freezer all this time.Episode VII. Since then, everything has been made by molecular biology, and that’s due to this man,here, Douglas Prasher at the center of the photograph, who in 1992 published the cloning of the genefor GFP. And that was a long struggle, he didn’t know what to look for, and he could not predict thatit would become green fluorescent. In fact, that was a major worry that there was no precedent for aprotein that, made by a biological organism, would absorb blue light and fluoresce green. We didn’tknow whether it needed any co-factors.

Normally, in many cases, when biology does this sort ofremarkable thing with light, it uses small molecule co-factors. You’re seeing me, for example, notbecause the proteins in your eye directly absorb the light through the protein, but rather because theproteins bind retinal, the pigment that we have to get from other sources, and that binds into theprotein to make the functional protein. And for all we knew, the fear was that maybe this proteinused another co-factor or the chromophore structure that Shimamura had proposed might take many288enzymes to generate out of the protein. But, Marty Chalfie, who is shown here on the right side ofthe picture, then got to the clone from the gene, from Prasher, as I did as well, but Marty was firstand proved that just that gene alone was self-sufficient; in other words, if you took that gene andexpressed it in other organisms, and he did it in E.Coli and in worms, then you would generatefluorescence.

And that proved that GFP was self-sufficient. It knew how to make its ownchromophore out of its own guts. Fred Tsuji, not show here, also did it very very soon after Chalfieindependently.Episode VIII. So, we now know that the chromophore structure is something like this structure here,at the right, but it had to come from the protein structure shown here, where just at this stage stillamino acids.

There’s serine, tyrosine and glycine that somehow have to be modified, and we have tointroduce through a various number of steps, which people are still now somewhat debating, exactlyhow it happens. We generated an extra double bond, we generate a ring and so on. And all of thisdoes require oxygen: if you do not have oxygen in the atmosphere, when this protein is growing, itcannot become fluorescent or doesn’t even absorb light – let alone become fluorescent. So, theoxygen is essential, and the only creatures which are biologically incapable of using GFP, assumingwe can get the DNA, the gene for GFP, they would be organisms that cannot tolerate oxygen at anystage in their life cycle.

In other words, obligate anaerobes. But they are limitation, you do needoxygen.Episode IX. So, then, this may seem to be a completely unprecedented reaction, but it turns out thatthe crucial first step is the attack of a backbone NH from glycine-67 onto the amide bond of serine65. And this is a very strange reaction, you might think. Since when does one amide attack another?But in fact, it turns out to be fairly similar to a known reaction in biochemistry, where the side chainamide of glycine attacks the side chain amide of asparagine. And that is promoted by bringing themclosely into a chain, and it requires the special properties of glycine that it can curl up; and also thatit’s relatively unencumbered in its NH-group. And it’s interesting that of all the fluorescent proteinsthat have ever been discovered the most conserved residue is not the one that contributes the mostatoms to the chromophore nor this other one that gets attacked, but rather glycine-67 – every knownfluorescent protein has glycine-67 in it.

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