諾獎青睞交叉學科？說說拿到諾貝爾化學獎的物理學家……

海歸學者發起的公益學術平台

分享信息，整合資源

交流學術，偶爾風月

北京時間10月4日下午5點45分，瑞典皇家科學院宣布將2017年諾貝爾化學獎授予：來自瑞士洛桑大學的Jacques Dubochet，美國哥倫比亞大學的Joachim Frank以及英國劍橋分子生物學實驗室的Richard Henderson，表彰他們「開發在溶液中測定生物分子結構的高解析度冷凍電鏡」。很多人發現，這次獲得化學獎的三位學者似乎又是物理學家，這再次體現出了諾貝爾獎對於交叉學科的重視。

首先，讓我們看看近年中獲得諾貝爾化學獎的物理學者。

1998年：DFT之父

Walter Kohn

1998年度諾貝爾化學獎的頒布，向人們展示了數學、物理和化學學科的交叉和融合取得的重大成果。美國物理學家瓦爾特·科恩（Walter Kohn）和英國數學家約翰·波普（John Pople）以物理和數學工具，發展了量子化學理論和計算方法，在化學領域取得了驕人成就。通過以科恩和波普為代表的量子化學工作者的不斷努力，今天，量子化學無疑成為化學工作者最有用的工具之一。磁共振成像技術（MRI）的發明實質上是物理學與醫學的結合，也是交叉學科能產生豐富成果的有力證明。這種能精確觀察人體內部器官而又不造成傷害的影像技術，對於醫療診斷、治療及其檢查至關重要。

遺憾的是，瓦爾特·科恩於2016年4月19日在加利福尼亞家中因癌症逝世，享年93歲。

2014年：超高解析度熒光顯微鏡

William E. Moerner

關於2014年的諾貝爾化學獎，不少人也有疑問：幾位擁有物理學博士學位背景的人，發明的是在生命科學領域應用的激光物理技術，為什麼會獲得諾貝爾化學獎？甚至有些人還這樣認為：對超高分辨顯微技術看起來沒有那麼直接的貢獻的William E. Moerner，為什麼也是三位獲獎者之一？

其實，超高分辨熒光顯微成像技術之所以能夠繞過所謂光學衍射極限的物理限制——200納米，是建立在利用特殊熒游標記分子的光化學和光物理性質基礎之上的科學發展，其重要的科學基礎是單分子光譜和單分子顯微技術。Moerner對凝聚相中單分子光譜技術和綠熒光蛋白特殊的光學開關性質研究貢獻卓著，他獲得此項諾貝爾化學獎，自然當之無愧。

2017年：冷凍電鏡

何為冷凍電鏡？水結冰後會阻礙原先在溶液狀態下快速的物質交換與擴散，低溫使得化學過程速率降低：絕大多數代謝過程變得非常非常慢以至於我們難以察覺，這個技術叫做冷凍固定術（Cryo-fixation）。應用冷凍固定術，在低溫下使用透射電子顯微鏡觀察樣品的顯微技術，就叫做冷凍電鏡（CryoEM）。冷凍電鏡是重要的結構生物學研究方法，與之地位相當的另兩種技術是X射線晶體學（X-raycrystallography）和核磁共振（NMR），這些方法都是為了獲得生物大分子的結構以了解其功能。冷凍電鏡，就是把樣品凍起來然後保持低溫放進顯微鏡裡面，高度相干的電子作為光源從上面照下來，透過樣品和附近的冰層，受到散射。我們再利用探測器和透鏡系統把散射信號成像記錄下來，最後進行信號處理，得到樣品的結構。

而今天的三位諾貝爾化學獎獲得者有著怎樣的背景呢？

雅克·迪波什（Jacques Dubochet），1942年生於瑞士，1973年博士畢業於日內瓦大學和瑞士巴塞爾大學，瑞士洛桑大學生物物理學榮譽教授。迪波什博士領導的小組開發出真正成熟可用的快速投入冷凍制樣技術製作不形成冰晶體的玻璃態冰包埋樣品，隨著冷台技術的開發，冷凍電鏡技術正式推廣開來。

約阿基姆·弗蘭克（Joachim Frank），德裔生物物理學家，現為哥倫比亞大學教授。他因發明單粒子冷凍電鏡（cryo-electron microscopy）而聞名，此外他對細菌和真核生物的核糖體結構和功能研究做出重要貢獻。弗蘭克2006年入選為美國藝術與科學、美國國家科學院兩院院士。2014年獲得本傑明·富蘭克林生命科學獎。

理查德·亨德森（Richard Henderson），蘇格蘭分子生物學家和生物物理學家，他是電子顯微鏡領域的開創者之一。1975年，他與Nigel Unwin通過電子顯微鏡研究膜蛋白、細菌視紫紅質，並由此揭示出膜蛋白具有良好的機構，可以發生α-螺旋。近年來，亨德森將注意力集中在單粒子電子顯微鏡上，即用冷凍電鏡確定蛋白質的原子解析度模型。

簡而言之，Richard Henderson開創了二維電子晶體學三維重構技術，奠定了基本理論；Joachim Frank發明了冷凍電鏡單粒子三維重構技術；Jacques Dubochet則在早期實驗方面做出了重要貢獻。

未來科研的方向

其實，回顧百餘年來的諾貝爾獎，近半數獲獎者屬於交叉學科。尤其在近幾十年來，這一現象屢見不鮮。這對於新時代學者有著相當的借鑒意義。不同的學科彼此交叉綜合，有利於科學上的重大突破，培育新的生長點，乃至新學科的產生。無疑，交叉學科研究是科學發展的主要方向，也將越來越是諾貝爾獎的眷顧對象。

以下內容源自諾貝爾獎官方網站

Over the last few years, numerous astonishing structures of life』s molecular machinery have filled the scientific literature (figure 1): Salmonella』s injection needle for attacking cells; proteins that confer resistance to chemotherapy and antibiotics; molecular complexes that govern circadian rhythms; light-capturing reaction complexes for photosynthesis and a pressure sensor of the type that allows us to hear. These are just a few examples of the hundreds of biomolecules that have now been imaged using cryo-electron microscopy (cryo-EM).

When researchers began to suspect that the Zika virus was causing the epidemic of brain-damaged newborns in Brazil, they turned to cryo-EM to visualise the virus. Over a few months, threedimensional (3D) images of the virus at atomic resolution were generated and researchers could start searching for potential targets for pharmaceuticals.

Figure 1. Over the last few years, researchers have published atomic structures of numerous complicated protein complexes. a. A protein complex that governs the circadian rhythm. b. A sensor of the type that reads pressure changes in the ear and allows us to hear. c. The Zika virus.

Jacques Dubochet, Joachim Frank and Richard Henderson have made ground-breaking discoveries that have enabled the development of cryo-EM. The method has taken biochemistry into a new era, making it easier than ever before to capture images of biomolecules.

Pictures – an important key to knowledge

In the first half of the twentieth century, biomolecules – proteins, DNA and RNA – were terra incognita on the map of biochemistry. Scientists knew they played fundamental roles in the cell, but had no idea what they looked like. It was only in the 1950s, when researchers at Cambridge began to expose protein crystals to X-ray beams, that it was first possible to visualise their wavy and spiralling structures.

In the early 1980s, the use of X-ray crystallography was supplemented with the use of nuclear magnetic resonance (NMR) spectroscopy for studying proteins in solid state and in solution. This technique not only reveals their structure, but also how they move and interact with other molecules.

Thanks to these two methods, there are now databases containing thousands of models of biomolecules that are used in everything from basic research to pharmaceutical development. However, both methods suffer from fundamental limitations. NMR in solution only works for relatively small proteins. X-ray crystallography requires that the molecules form well-organised crystals, such as when water freezes to ice. The images are like black and white portraits from early cameras – their rigid pose reveals very little about the protein』s dynamics.

Also, many molecules fail to arrange themselves in crystals, which caused Richard Henderson to abandon X-ray crystallography in the 1970s – and this is where the story of 2017』s Nobel Prize in Chemistry begins.

Problems with crystals made Henderson change track

Richard Henderson received his PhD from the bastion of X-ray crystallography, Cambridge, UK. He used the method for imaging proteins, but setbacks arose when he attempted to crystallise a protein that was naturally embedded in the membrane surrounding the cell.

Membrane proteins are difficult to manage. When they are removed from their natural environment – the membrane – they often clump up into a useless mass. The first membrane protein that Richard Henderson worked with was difficult to produce in adequate amounts; the second failed to crystallise. After years of disappointment, he turned to the only available alternative: the electron microscope.

It is open to discussion whether electron microscopy really was an option at this time. Transmission electron microscopy, as the technique is called, works more or less like ordinary microscopy, but a beam of electrons is sent through the sample instead of light. The electrons』 wavelength is much shorter than that of light, so the electron microscope can make very small structures visible – even the position of individual atoms.

In theory, the resolution of the electron microscope was thus more than adequate for Henderson to obtain the atomic structure of a membrane protein, but in practice the project was almost impossible. When the electron microscope was invented in the 1930s, scientists thought that it was only suitable for studying dead matter. The intense electron beam necessary for obtaining high resolution images incinerates biological material and, if the beam is weakened, the image loses its contrast and becomes fuzzy.

In addition, electron microscopy requires a vacuum, a condition in which biomolecules deteriorate because the surrounding water evaporates. When biomolecules dry out, they collapse and lose their natural structure, making the images useless.

Almost everything indicated that Richard Henderson would fail, but the project was saved by the special protein that he had chosen to study: bacteriorhodopsin.

The best so far was not good enough for Henderson

Bacteriorhodopsin is a purple-coloured protein that is embedded in the membrane of a photosynthesising organism, where it captures the energy from the sun』s rays. Instead of removing the sensitive protein from the membrane, as Richard Henderson had previously tried to do, he and his colleague took the complete purple membrane and put it under the electron microscope. When the protein remained surrounded by the membrane it retained its structure; they covered the sample』s surface with a glucose solution that protected it from drying out in the vacuum.

The harsh electron beam was a major problem, but the researchers made use of how the bacteriorhodopsin molecules are packed in the organism』s membrane. Instead of blasting it with a full dose of electrons, they had a weaker beam flow through the sample. The image』s contrast was poor and they could not see the individual molecules, but they used the fact that the proteins were regularly packed and oriented in the same direction. When all the proteins diffract the electron beams in an almost identical manner, they were able to calculate a more detailed image based on the diffraction pattern – they used a similar mathematical approach to that used in X-ray crystallography.

At the next stage, the researchers turned the membrane under the electron microscope, taking pictures from many different angles. This way, in 1975 it was possible to produce a rough 3D model of bacteriorhodopsin』s structure (figure 2), which showed how the protein chain wiggled through the membrane seven times.

Figure 2. The first rough model of bacteriorhodopsin, published in 1975. Image from Nature 257: 28-32

It was the best picture of a protein ever generated using an electron microscope. Many people were impressed by the resolution, which was 7 ?ngstr?m (0.0000007 millimetres), but this was not enough for Richard Henderson. His goal was to achieve the same resolution as that provided by X-ray crystallography, about 3 ?ngstr?m, and he was convinced that electron microscopy had more to give.

Henderson produces the first image at atomic resolution

Over the following years, electron microscopy gradually improved. The lenses got better and cryotechnology developed (we will return to this), in which the samples were cooled with liquid nitrogen during the measurements, protecting them from being damaged by the electron beam.

Richard Henderson gradually added more details to the model of bacteriorhodopsin. To get the sharpest images he travelled to the best electron microscopes in the world. They all had their weaknesses, but complemented each other. Finally, in 1990, 15 years after he had published the first model, Henderson achieved his goal and was able to present a structure of bacteriorhodopsin at atomic resolution (figure 3).

Figure 3. In 1990, Henderson presented a bacteriorhodopsin structure at atomic resolution.

He thereby proved that cryo-EM could provide images as detailed as those generated using X-ray crystallography, which was a crucial milestone. However, this progress was built upon an exception: how the protein naturally packed itself regularly in the membrane. Few other proteins spontaneously order themselves in this way. The question was whether the method could be generalised: would it be possible to use an electron microscope to generate high-resolution 3D images of proteins that were randomly scattered in the sample and oriented in different directions? Richard Henderson believed it would be, while others thought this was a utopia.

On the other side of the Atlantic, at the New York State Department of Health, Joachim Frank had long worked to find a solution to just that problem.

In 1975, he presented a theoretical strategy where the apparently minimal information found in the electron microscope』s two-dimensional images

could be merged to generate a high-resolution, three-dimensional whole. It took him over a decade to realise this idea.

Frank refines image analysis

Joachim Frank』s strategy (figure 4) built upon having a computer discriminate between the traces of randomly positioned proteins and their background in a fuzzy electron microscope image. He developed a mathematical method that allowed the computer to identify different recurring patterns in the image. The computer then sorted similar patterns into the same group and merged the information in these images to generate an averaged, sharper image. In this way he obtained a number of high-resolution, two-dimensional images that showed the same protein but from different angles. The algorithms for the software were complete in 1981.

Figure 4

The next step was to mathematically determine how the different two-dimensional images were related to each other and, based on this, to create a 3D image. Frank published this part of the image analysis method in the mid-1980s and used it to generate a model of the surface of a ribosome, the gigantic molecular machinery that builds proteins inside the cell.

Joachim Frank』s image processing method was fundamental to the development of cryo-EM. Now we』ll jump a few years back in time – in 1978, at the same time as Frank was perfecting his computer programs, Jacques Dubochet was recruited to the European Molecular Biology Laboratory in Heidelberg to solve another of the electron microscope』s basic problems: how biological samples dry out and are damaged when exposed to a vacuum.

Dubochet makes glass from water

In 1975, Henderson used a glucose solution to protect his membrane from dehydrating, but this method did not work for water-soluble biomolecules. Other researchers had tried freezing the samples because ice evaporates more slowly than water, but the ice crystals disrupted the electron beams so much that the images were useless.

The vaporising water was a major dilemma. How- ever, Jacques Dubochet saw a potential solution: cooling the water so rapidly that it solidified in its liquid form to form a glass instead of crystals. A glass appears to be a solid material, but is actu- ally a fluid because it has disordered molecules.

Dubochet realised that if he could get water to form glass – also known as vitrified water – the electron beam would diffract evenly and provide a uniform background.

Initially, the research group attempted to vitrify tiny drops of water in liquid nitrogen at –196°C, but were successful only when they replaced the nitrogen with ethane that had, in turn, been cooled by liquid nitrogen. Under the microscope they saw a drop that was like nothing they had seen before. They first assumed it was ethane, but when the drop warmed slightly the molecules sud- denly rearranged themselves and formed the fami- liar structure of an ice crystal. It was a triumph – particularly as some researchers had claimed it was impossible to vitrify water drops. We now believe that vitrified water is the most common form of water in the universe.

A simple technique for contrast

After the breakthrough in 1982, Dubochet』s research group rapidly developed the basis of the technique that is still used in cryo-EM (figure 5). They dissolved their biological samples – initially different forms of viruses – in water. The solution was then spread across a fine metal mesh as a thin film. Using a bow-like construction they shot the net into the liquid ethane so that the thin film of water vitrified.

Figure 5

In 1984, Jacques Dubochet published the first images of a number of different viruses, round and hexa- gonal, that are shown in sharp contrast against the background of vitrified water. Biological material could now be relatively easily prepared for electron microscopy, and researchers were soon knocking on Dubochet』s door to learn the new technique.

From blobology to revolution

The most important pieces of cryo-EM were thus in place, but the images still had poor resolution. In 1991, when Joachim Frank prepared ribosomes using Dubochet』s vitrification method and analysed

the images with his own software, he obtained a 3D structure that had a resolution of 40 ?. It was an amazing step forward for electron microscopy, but the image only showed the ribosome』s conto- urs. Frankly, it looked like a blob and the image did not even come close to the atomic resolution of X-ray crystallography.

Because cryo-EM could rarely visualise anything other than an uneven surface, the method was sometimes called 「blobology」. However, every nut and bolt of the electron microscope has gradually been optimised, greatly due to Richard Henderson stubbornly maintaining his vision that electron microscopy would one day routinely provide images that show individual atoms. Resolution has improved, ?ngstr?m by ?ngstr?m, and the final technical hurdle was overcome in 2013, when a new type of electron detector came into use (figure 6).

Figure 6. The electron microscope』s resolution has radically improved in the last few years, from mostly showing shapeless blobs to now being able to visualise proteins at atomic resolution. Image: Martin H?gbom.

Every hidden corner of a cell can be explored

Now the dream is reality, and we are facing an explosive development within biochemistry. There are a number of benefits that make cryo-EM so revolutionary: Dubochet』s vitrification method is relatively easy to use and requires a minimal sample size. Due to the rapid cooling process, biomolecules can be frozen mid-action and researchers can take image series that capture different parts of a process. This way, they produce 『films』 that reveal how proteins move and interact with other molecules.

Using cryo-EM, it is also easier than ever before to depict membrane proteins, which often function as targets for pharmaceuticals, and large molecular complexes. However, small proteins cannot be studied with electron microscopy, but they can be visualised using NMR spectroscopy or X-ray crystallography.

After Joachim Frank presented the strategy for his general image processing method in 1975, a researcher wrote: 「If such methods were to be perfected, then, in the words of one scientist, the sky would be the limit.」

Now we are there – the sky is the limit. Jacques Dubochet, Joachim Frank and Richard Henderson have, through their research, brought 「the greatest benefit to mankind.」 Each corner of the cell can be captured in atomic detail and biochemistry is all set for an exciting future.

喜歡這篇文章嗎？立刻分享出去讓更多人知道吧！

本站內容充實豐富，博大精深，小編精選每日熱門資訊，隨時更新，點擊「搶先收到最新資訊」瀏覽吧！

請您繼續閱讀更多來自 知社學術圈 的精彩文章: