Novartis Foundation Symposium 224

RHODOPSINS AND PHOTOTRANSDUCTION

1999

JOHN WILEY & SONS, LTD

Chichester New York . Weinheim . Brisbane . Singapore . Toronto

RHODOPSINS AND PHOTOTRANSDUCTION

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Novartis Foundation Symposium 224

RHODOPSINS AND PHOTOTRANSDUCTION

1999

JOHN WILEY & SONS, LTD

Chichester New York . Weinheim . Brisbane . Singapore . Toronto

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Novartis Foundation Symposium 224 ix+306 pages, 74 figures, 11 tables

Library ofcongress Ca~aloging--in-Publication Data Rhodopsins and phototransduction.

p. cm. - (Novartis Foundation symposium ; 224) Includes bibliographical references and index. ISBN 0-471-98827-8 (alk. paper) 1. Rhodopsin Congresses. I. Symposium on Rhodopsins and

11. Series. Phototransduction (1998: Kyoto, Japan). QP671.V5R48 1999 573.8'8459-dc21 99-37597

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Contents

The Novartis Foundation in collaboration with the Novartis Foundation (Japan) for the

Jjmposium on Rhodopsins andphototransduction, held in Kyoto International Conference Hal/,

This gmposium is held in commemoration ojthe late Projessor George Wald, and is based on a

Promotion of Science

Rjoto, Japan, 2 6 2 8 October 1998

proposalmade Ly Projessor?iiruyoshi~awa

Editors: Ikuo Takeuchi and Gregovl Bock (O,yani&, andJamie A. Goode

Toru Yoshizawa Chairman’s introduction 1

Ruth Hubbard and Elijah Wald George Wald memorial talk 5 Discussion 18

James K. Bowmaker The ecology of visual pigments 21 Discussion 31

General discussion I Vertebrate ancient (VA) opsin: a new vertebrate photopigment family 36

Fumio Tokunaga, Osamu Hisatomi, Takunori Satoh,Yuki Taniguchi, Shinji Matsuda,Yoshikazu Imanishi, Hanayo Honkawa,YusukeTakahashi, Yuko Kobayashi, MasaoYoshida and Yasuo Tsukahara pigments and related molecules 44 Discussion 52

Evolution of visual

Gebhard F. X. Schertler Structure of rhodopsin 54 Discussion 6 6

Richard A. Mathies Photons, femtoseconds and dipolar interactions: a molecular pkture of the primary events in vision Discussion 84

70

General discussion I1 90

Willem J. DeGrip, Frank DeLange, Corn6 H. W. Klaassen, Peter J. M.Verdegem, Stacie Wallace-Williams, Alain F. L. Creemers,Vladislav Bergo, Petra H. M. Bovee, Jan Raap, Kenneth J. Rothschild, Huub J. M. DeGroot and Johan Lugtenburg chromophore 102 Discussion 1 18

Photoactivation of rhodopsin: interplay between protein and

V

vi CONTENTS

Steven W. Lin and Thomas P. Sakmar Colour tuning mechanism of visual pigments 124 Discussion 135

Yoshinori Shichida and Hiroo Imai Amino acid residues controlling properties and functions of rod and cone visual pigments Discussion 153

142

Klaus Peter Hofmann Signalling states of photoactivated rhodopsin 158 Discussion 175

General discussion 111 181

Krzysztof Palczewski, Christophe L. M. J.Verlinde and Franqoise Haeseleer Molecular mechanism of visual transduction Discussion 204

191

Satoru Kawamura phosphorylation 208 Discussion 21 8

Calcium-dependent regulation of rhodopsin

Hiroyuki Matsumoto, Esther S. Kahn and Naoka Komori

225

The emerging role of mass spectrometry in molecular biosciences: studies of protein phosphorylation in fly eyes as an example Discmion 244

Robert S. Molday, Renk Warren, Chris Loewen and Laurie Molday GMP-gated channel and peripherin/rds-rom-1 complex of rod cells Discussion 261

Cyclic 249

Daisuke Kojima and Yoshitaka Fukada Non-visual photoreception by a variety of vertebrate opsins 265 Discussion 279

Final discussion 283

'EiruYoshizawa Chairman's summing-up 291

Index of contributors 297

Subject index 299

Participants

James K. Bowmaker Department of Visual Science, Institute of Ophthalmology, University College London, Bath Street, London EClV OEL, UK

Willem J. DeGrip Signalling, University of Nijmegen, PO Box 9101,6500 H B Nijmegen, The Netherlands

Department of Biochemistry, FMW-160, Institute of Cellular

John E. Dowling University Biological Laboratories, 16 Divinity Avenue, Cambridge, MA 02138, USA

Department of Molecular and Cellular Biology, Harvard

Russell Foster Sir Alexander Fleming Building, Department of Biology (Rm 549, Imperial College of Science, Technology and Medicine, London SW7 2A2, UK

Yoshitaka Fukada Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo 7- 3-1, Bunkyo ku, Tokyo 113-0032, Japan

Klaus Peter Hofmann Institut fur Medizinische Physick und Biophysik, Charite-Humboldt University, Schumannstrasse 20-21, D-10098 Berlin, Germany

Ruth Hubbard Harvard University Biological Laboratories, 16 Divinity Avenue, Cambridge, MA 02138, USA

Yasushi Imamoto (Nuvurtis Foundutiun Bursar) Graduate School of Materials Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan

Toshiaki Kakitani Department of Physics, Graduate School of Science, Nagoya University, Furoucho, Chigusaku, Nagoya 464-8602, Japan

vii

viii PARTICIPANTS

Hideki Kandori Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

U. Benjamin Kaupp Forschungszentrum Jiilich, Institut fur Biologische Informationsverarbeitung, 52425 Jiilich, Germany

Satoru Kawamura Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan

H. Gobind Khorana Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139-4307, USA

Richard A. Mathies Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA

Hiroyuki Matsumoto Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, PO Box 26901, Oklahoma City, OK 73104, USA

Robert S. Molday Department of Biochemistry and Molecular Biology, University of British Columbia, Faculty of Medicine, 2146 Health Sciences Mall, Vancouver BC, CanadaV6T 123

Tadashi Nakamura Department of Applied Physics and Chemistry, University of Electro-Communications, Chofu, Tokyo 182-8585, Japan

Krzysztof Palczewski Depart,ment of Ophthalmology, University of Washington, PO Box 356485, Seattle,WA 98195-6485, USA

Thomas P. Sakmar The Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY 10021, USA

Gebhard F. X. Schertler MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

Yoshinori Shichida Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

IkuoTakeuchi Novartis Foundation (Japan) for the Promotion of Science, 10-66 Miyuki-Cho,Takarazuka 665, Japan

PARTICIPANTS 1x

FumioTokunaga Department of Earth and Space Science and Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560- 0043, Japan

Elijah Wald 39 Burnham Street, Somerville, MA 02144, USA

AkioYamazaki The Kresge Eye Institute, Departments of Ophthalmology and Pharmacology, Wayne State University, School of Medicine, 4717 St. Antoine Blvd, Detroit, MI 48202, USA

ToruYoshizawa (Chairman) Osaka Sangyo University, 3-1-1 Nakagaito, Daito-shi, Osaka 574-8530, Japan

Chairman’s introduction T6ru Yoshizawa

Osaka Sangyo University, 3- I - I Nakagaito, Daito-shi, Osaka 574-8530,]apun

In October 1998, the Novartis Foundation (UK) in collaboration with the Novartis Foundation (Japan) for the Promotion of Science jointly organized a symposium on Rhodopsins and phototransduction. It was a great pleasure for me to introduce the symposium dedicated to the memory of Professor George Wald, with Ruth Hubbard Wald and their son Elijah Wald both present. I would like to briefly describe why the symposium was held at this time in Kyoto. It was in April 1997 that I heard the sad news of Professor Wald’s death. At the ARVO (Association for Research in Vision and Ophthalmology) meeting that May, I met Professor John Dowling of Harvard University and promised him that I would organize a memorial symposium in Japan, because the 8th International Conference of Retinal Protein had been scheduled to be held in June 1998 at Awaji island, close to Kansai Airport. Shortly after, I was informed by Professor Ikuo Takeuchi of the Novartis Foundation (Japan) that they wanted me to serve as the chairman of this symposium with the provisional title of ‘Rhodopsin and vision’ and, later, that the symposium was going to be held in the autumn in Kyoto. Since it may safely be said that all the research achievements in this field are based on the work of George Wald, and bearing in mind he had a great affection for Kyoto, an old capital of Japan, I thought it would be appropriate that this Novartis Foundation symposium should become the memorial symposium.

As you may know, George Wald was awarded a Nobel Prize in 1967. Though his achievements will be described in detail by Professor Ruth Hubbard in the first paper of this book, I would like to touch on them briefly here. His accomplishments were summarized in the presentation speech by Professor C. G. Bernhard, a member of the Nobel Committee. Here are the last few sentences of his qeech, in italics, with my comments underneath.

‘Professor Wuld. W i t h a deep biologicalins@andagreat biochemicalskill , . . ’

In fact, he had a wide knowledge of science, from psychology to quantum chemistry, and studied mainly vision using various techniques (e.g. psychophysical, morphological, electrophysiological, biochemical and spectroscopical techniques) on the one hand, and on the other hand enunciated his scientific philosophy from origin of life to evolution of consciousness.

1

2 YOSHIZAWA

'. . gou have successjXb identzfed visaalpigments and theirprecursors. As a ly-product you were able t o describe the absorption spectra ofthe different opes of cones serving colour vision. . . .'

He discovered vitamin A in the retina, and retinal as a photoproduct of rhodopsin. Later he found all-trans-retinal as the photoproduct and 11 -&retinal as the chromophore of rhodopsin. He also identified many new visual pigments from various animals, for example, porphyropsin (with 3-dehydroretinal as the chromaphore) in freshwater fish and chicken iodopsin (as the first extracted cone visual pigment). Using microspectrophotometry and psychophysical techniques, he measured three types of cone pigment and established the molecular basis of human trichromatic theory. Based on the chromophoric retinal, he presented a phylogenetic tree of visual pigments.

' . . . Yow most important discove9 of the primary molecular reaction t o light in the eye represents a dramatic advance in vision since it p l g s the role of a triger in the photoreceptors of all living animals.'

Professor Wald described many intermediates of the photobleaching process of rhodopsin by low temperature spectrophotometry (Batho, Lumi, Meta I, Meta I1 and Meta I11 rhodopsins) and provided a substantial basis for the photoisomerization hypothesis in vision (the only action of light in vision is to isomerize the chromophore of visual pigment) by proposing that the change from rhodopsin to bathorhodopsin is due to the photoisomerization of chromophoric retinal from the 11 -cis form to a twisted all-trans one. He also drew his inference from several lines of evidence that the conversion from metarhodopsin I to I1 would be a crucial step of triggering of visual excitation. In addition, he proposed a biochemical cascade model for visual transduction after the example of blood clotting.

Since George Wald received the Nobel Prize, the analyses of the structure and function of rhodopsin molecule and the biochemical amplification mechanism in rod outer segment have been extensively studied by physical, chemical, biochemical and molecular biological techniques. In particular, the rapid progress in this field that has taken place over the last decade has been amazing. We now understand an outline of the main routes of phototransduction from absorption of a photon by a rhodopsin molecule to generation of the receptor potential as in the following.

Rhodopsin is a membrane-embedded protein composed of seven transmembrane helices and a member of the G protein-coupled receptor family, among which rhodopsin is the first one whose tertiary structure has been investigated. On absorbing a photon, the chromophoric retinal isomerizes

CHAIRMAN’S INTRODUCTION 3

from 11-cis to a twisted all-trans configuration within 1 ps, resulting in formation of the first photoproduct, photorhodopsin. This ultrafast photo- isomerization is the most rapid switching device so far known. Subsequent thermal reactions induce stepwise conformational changes of opsin, resulting in the formation of several intermediates, each of which has a specific absorption spectrum, and finally produce an enzymatically active intermediate, metarhodopsin 11. One molecule of metarhodopsin I1 activates several hundreds molecules of G protein (transducin). This is the first amplification step in the phototransduction cascade. The activated G protein in turn activates a cGMP- phosphodiesterase, which hydrolyses several hundred molecules of cGMP. This is the second step of amplification in phototransduction. The decrease in concentration of cytosolic cGMP causes liberation of cGMP from the cation channels in the plasma membrane, resulting in a closure of the channel. Thus the hyperpolarizing receptor potential is generated from rod outer segment. Phosphorylation of metarhodopsin I1 by rhodopsin kinase and binding of arrestin to the phosphorylated rhodopsin may stop the activation of transducin by metarhodopsin 11. The phototransduction system would be modulated by several proteins such as S-modulin (recoverin), guanylate cyclase activating protein (GCAP), phosducin and calmodulin in a Ca2+-dependent manner. These modulations would induce light and dark adaptations. On the basis of the many experimental results obtained from the rhodopsin molecule and its related proteins, we shall soon understand the general mechanism of the signal transduction mediated by the G protein-coupled receptor family.

In organizing this symposium, we have attempted to take a multi- disciplinary approach to the study of rhodopsins and phototransduction. In the first session, we will look at the divergence of visual pigments. Recent findings have revealed that many diurnal vertebrates have visual systems with one type of rhodopsin and four types of cone pigments. As possible topics for general discussion, I would like to suggest the following subjects. Which type of visual pigment is close to the ancestor type of vertebrate visual pigments? Which phylogenetic tree of visual pigments is currently most reliable?

In the second session, the papers will describe the tertiary structure and the photoreaction of rhodopsin studied by using a variety of physical techniques, for example, cryoelectron microscopy of two-dimensional crystals of rhodopsin, femtosecond laser photolysis, and Resonance Raman, Fourier transform infrared and solid-state NMR spectroscopies with the help of isotope-labelled rhodopsins. In the general discussion, it is expected that the change of the chromophoreamino acid residue interaction from femtoseconds to seconds after the absorption of light will be discussed and an up-to-date model for the photobleaching process of rhodopsin will be presented.

4 YOSHIZAWA

The third session covers the molecular biology of visual pigments. Studies on physiological functions of visual pigment including the colour tuning mechanism, differences in structure and function between rod and cone pigments, and signalling states of metarhodopsin I1 will be reported on the basis of chromophoreamino acid residue and protein-protein interactions. In the general discussion, these physiological functions should be discussed in connection with the retinal binding pocket and the G protein binding site, which will also be discussed in the second session.

In the fourth session, various Ca2+-binding proteins, including S-modulin (recoverin) and GCAP will be described and their physiological functions will be discussed. In the general discussion, the current status of studies on the role of Ca2+ in phototransduction and its modulation will be covered.

In the final session, studies on protein phosphorylation in fly eyes, the cGMP- gated channel, peripherin and non-visual photoreception will be presented. The possible topics for the general discussion include the significance of post- translational modification in visual cells, and a comparison of transduction mechanisms between visual and non-visual photoreceptor cells including other sensory receptor cells.

In each of the topics I have just touched on, I feel strongly that George Wald built up the base for interdisciplinary study on rhodopsins and phototransduction, and his memory will remain with us.

George Wald memorial talk Ruth Hubbard and Elijah Wald*

Biological Laboratories, Harvard University, 16 Divinity A venue, Cambridge, M A 02 138, U S A

A bstruct. George Wald was born in 1906 in New York City to immigrant parents. An early and voracious reader, he soon developed a wide range of interests and entered New York University as a pre-law student, the first in his family to attend college. Shortly shifting to pre-medicine, he graduated college in biology. For graduate work, he joined the laboratory of Selig Hecht, a pioneer in vision research, at Columbia University. In 1932, four months before Hitler came to power, George went to Berlin to do postdoctoral work in the laboratory of Otto Warburg and there found vitamin A in the retina. This launched his life-long explorations of the molecular basis of vision for which he received the Nobel Prize in Physiology or Medicine in 1967. During the 1960s, George became increasingly involved in anti-war and anti-nuclear activities, writing and travelling widely, including multiple trips to commemorations of the bombings of Hiroshima and Nagasaki sponsored by Japanese colleagues. He considered these activities part of being a biologist, someone concerned with life. In his final years, he turned to questions about consciousness, writing and speaking about ‘Life and Mind in the Universe’.

1999 RhodopJins and phototransduction. Wihy, Cbichester (Novurtis Foundation Symposium 224) p 5-20

1.

George Wald was born in New York City, on November 18, 1906. His parents were both immigrants; his mother from Germany, his father from Poland. He grew up in Brooklyn, New York, in a working-class neighbourhood. His scientific bent manifested itself first in an interest in electricity, which was a very exciting subject at the time for a kid. He became a regular reader of a magazine called The Electrical Experimenter and, with his friend Freddie Fisher, who lived across the backyards from him, began doing experiments. They built small electromagnets, a telephone with which they could talk back and forth across the

*In their live performance, Elijah Wald delivered parts I and 111, Ruth Hubbard part 11. The same is true of this paper.

5

6 HUBBARD & WALD

backyards, and a crystal detector radio. George would remember that radio as bringing the first great victory of his life. He was always very bothered by the fact that he was not good at sports; one year, just after he had entered high school, he was able to build a crystal detector radio and hear the World Series in his home, and all the kids from the neighbourhood came in to listen. This was a heroic moment for him.

George went to Manual Training High School, a technical high school that trained students for jobs where they worked with their hands, rather than for intellectual careers. In later years, he always took great pleasure and pride in the fact that he had gone to a technical high school and felt that this had been extremely useful to him. He was proud that, when he needed some unusual apparatus to be made, he could make the pattern and contribute to the building of the equipment and understand how it worked.

At this point, both he and Freddie Fisher were planning to be electrical engineers when they grew up. The way that ended was that Freddie Fisher’s father worked as a night watchman for Western Electric, in New Jersey, and he arranged for them, as budding electrical engineers, to take a tour of the offices there. George recalled that, at a moment in the tour, the person who was guiding them opened a door to an office that covered at least an acre, full of desks so tightly packed that you could barely squeeze between them, and the guide waved his had and said, ‘These are our electrical engineers.’ Then they went down the hall to another door, and there was another acre-sized office, and the guide waved his hand and said, ‘And these are more of our electrical engineers.’ As George would say to finish the story, ‘With that, I lost all ambition to become an electrical engineer.’

Along with his interest in electricity, George also had another important interest. With another friend, he had developed a small vaudeville act. They performed song and dance and comedy, and would go around to various Jewish community centres and give shows. His family was always looking for ways that the children would be able to make a living, and George’s success as a performer led his parents to say, ‘George talks good. He should be a lawyer.’ So, he went off to New York University (NYU) - chosen, he always said, because it was the first college on the subway line from Brooklyn- as a pre-law student.

George was the first member of his family to go to college, and it opened up a whole world to him. For the first time he came across classical music and literature, and he became a passionate lover of both. He even took to writing poetry, a very strange idea for someone from his neighbourhood. The broadening of his horizons continued outside college as well. For two consecutive summers, he shipped out on a passenger boat going to Buenos Aires and back. All of this was incredibly exciting for him. The world seemed full of possibilities, and he soon decided, ‘Law was not for me. It was an artificial, man-made thing and I needed to be able to get into something more substantial, more natural, more organic. As one was always

GEORGE WALD 7

pressed by the necessity of making a living, this turned me into a pre-medical student. ’

George did pre-medical studies at NYU, but by his senior year that had also begun to seem too prosaic a career. This time, the catalyst for his changing taste was a book by Sinclair Lewis called Arrowsmith. Arrowsmith was a young physician who was torn between, on the one hand, practising medicine and making money and, on the other, doing research. Research was presented as a noble quest for knowledge, rather than simply taking care of patients, and George became entranced with this idea. Therefore, by the time he graduated and entered Columbia University as a graduate student in zoology, he was interested only in doing research.

NYU had opened up all of these new worlds, but the level of the science instruction had not been particularly high. Columbia was a different story. In his first year, he took a genetics course with T. H. Morgan, and he also met the man who would be his mentor, Professor Selig Hecht.

11.

George entered Selig Hecht’s laboratory in 1927 as Hecht’s graduate student and research assistant. Hecht had been studying the light responses of the worm Ciona and the clam Mya as well as human vision. And he took great satisfaction in summing up these very different systems by assuming that, in all of them, a photosensitive substance S is decomposed by light into a product P and an accessory substance A, and that P + A , in light or dark, can recombine to regenerate S (Hecht 1920).

Hecht was mapping out a whole array of visual functions, but was not interested in these substances themselves, only in their physicochemical relationships. George admired Hecht tremendously. All his life, Hecht was his model, but by the time he left Hecht’s lab, he was dying to get his hands on the actual molecules. So, with a fellowship from the US National Research Council, in 1932, he went to the laboratory of Otto Warburg in Berlin, one of the great biochemists of the period, who had just won a Nobel Prize for discovering the Atmungsferment - what we now call cytochrome oxidase.

When George told Warburg he wanted to study rhodopsin, Warburg promptly asked, ‘What do you think rhodopsin is? Could it be an Atmungsferment?’ Warburg got one of his assistants to show George how to take out retinas, which was already exciting because in his four years in Hecht’s lab, George had never seen a retina. George put two batches of dark-adapted retinas into Warburg vessels, all under dim red light, and followed their oxygen uptake in the dark. He then exposed one batch to light and there was a big burst of oxygen uptake. All excited, he told Warburg and went back to confirm the experiment. He repeated

8 HUBBARD & WALD

it once, twice, three times - never again the slightest light response. Warburg took the news calmly: ‘Ah yes, Herr Wald,’ he said, ‘the better one’s technique, the harder it is to make discoveries.’ George then did some other experiments on the respiration of retinas, but, all the while, he kept wondering what rhodopsin might be.

Rhodopsin was discovered in 1876 in frog retinas by Franz Boll (1877) who realized that it must be a visual pigment. In the next two years, he and Willy Kiihne (1879), the professor of physiology at Heidelberg, described everything that was known about it up to this point: that it is responsible for the reddish purple colour of dark-adapted retinas, it bleaches in the light to yellowish orange and then more slowly to colourless, and that it is regenerated in the dark. Kiihne also brought rhodopsin into aqueous solution by means of bile salts and showed that it is a protein.

Thinking about it, George decided rhodopsin probably was not a haem protein, like the Atmungsferment, because the absorption spectrum was all wrong. More likely, it was a carotenoid pigment. So, he started reading about carotenoids and learned that they are fat-soluble and give strong colour reactions with antimony trichloride. He therefore shook up some retinas with chloroform, mixed the extract with an antimony chloride solution and it promptly turned blue, with an absorption band characteristic of vitamin A. Reading further, he realized that there was a considerable literature linking vitamin A deficiency and night blindness. No one yet knew how any vitamin functioned. Finding vitamin A in the retina suggested that it might participate directly in the visual process.

When George told Warburg, Warburg’s first reaction was: ‘Crystallize it; get a melting point.’ Well, it was years before anyone crystallized vitamin A, and not in the quantities you could get from frog retinas. But next Warburg said: ‘If you want to work on Atmungsferment, you are welcome to stay; but if you want to work on vitamin A, you had better go to Karrer.’ Paul Karrer, the great organic chemist in Zurich, had just worked out the structure of vitamin A and shown that it was half a molecule of /I-carotene, with a molecule of water inserted at the break, so a carotenoid alcohol. In fact, the previous summer Karrer had given a paper about this at the International Physiological Congress in Rome, which George had attended. But, since he was not yet interested in vitamin A, he had not heard Karrer’s talk.

Now, off he went to Zurich and collected thousands and thousands of cattle, sheep and pig eyes from slaughter houses. With the help of his wife Frances, he dissected their retinas and extracted them with fat solvents until he had collected enough material so Karrer could confirm that it was vitamin A (Wald 1935a). With that, George went back to Germany, this time to the laboratory of the biochemist Otto Meyerhof in Heidelberg, who had won a Nobel prize in 1922 for his work on the metabolism of muscle.

GEORGE WALD 9

But, this was a very different Germany from the one he had left only three months earlier, because on January 31, 1933, the day George had moved to Zurich, Hitler became Reichs Chancellor. It was now Nazi Germany. Meyerhof was Jewish, George was Jewish. One day, one of the lab assistants was picked up on the street and was not seen again-he had been a communist. The National Research Council wanted George out of Germany by the end of the summer. Not long afterwards, Meyerhof himself left for a professorship at the University of Pennsylvania.

While in Meyerhof s laboratory, George studied phosphates in the retina. But, a month before he was to return to the USA and while everyone else had gone off for their summer holidays, a shipment of 300 frogs arrived from Hungary.

Why from Hungary? Well, the Nazis were great animal lovers and had passed a law forbidding the killing of frogs - but only German frogs. (Aryan frogs, I suppose.) It was OK to kill Hungarian frogs. With everyone else on vacation, the assistant in charge was about to let the frogs go, but George asked to let him have them. With those frogs, he mapped out the rhodopsin cycle.

Extracting the retinas with fat solvents, he showed that rhodopsin and the orange intermediate of bleaching both release a yellow, previously unknown carotenoid, which he called retinene, and that, as the retinal colour disappears, retinene is replaced by vitamin A. With that, he wrote a note for Nature on retinene and vitamin A in the visual cycle and came back to the USA. He then examined other vertebrates and found the same cycle in their retinas (Wald 1935b). Kuhne and two later researchers, however, had described dark-adapted fish retinas as more purple than frog retinas. So, next summer in Woods Hole, George looked at several fish, but found that they, too, contained rhodopsin, retinene and vitamin A, like frogs (Wald 1936).

Thinking about it further, he realized that the earlier workers had been looking at freshwater fish and he at marine fish. That seemed an odd consideration, since marine and freshwater fish are very similar. But, as soon as he looked at some freshwater fishes, clearly their visual pigment was more purple, with its absorption maximum at longer wavelengths than rhodopsin. George therefore called it porphyropsin and showed that it bleaches to a different form of retinene and vitamin A, with their absorption maxima also displaced to longer wavelengths, which he called retinenez and vitamin A2 (Wald 1939).

That just raised the next question: what about euryhaline fishes, fishes that can go back and forth between fresh water and salt water? Extracting the retinas and livers of a number of species, George found that, irrespective of whether he caught them in fresh water or in the sea, their vitamin A, and therefore their visual pigment, went with the spawning environment: fishes that spawn in fresh water, like salmon or trout, have predominantly vitamin A2, those that spawn in the sea, like the eel, predominantly vitamin Al, the ordinary vitamin A (Wald 1941).

10 HUBBARD & WALD

FIG. 1. At the bench, circa 1937.

That raised the next question. If the kind of vitamin A animals have goes with the spawning environment, what about amphibia? They also spawn in fresh water, yet frogs were the animals in which rhodopsin was discovered. As luck would have it, one Sunday, George found his children playing by a pond full of tadpoles and little frogs in all stages of metamorphosis. And, with a selection of animals at different stages, he showed that the visual system of the frog changes from a pure A2 system in the earliest tadpoles, over mixtures of A2 and A,, to the pure Al system in adult frogs (Wald 1945-46).

George took special pleasure in the fact that, by confronting him with such puzzles, the visual system got him to think about the molecular transformations accompanying metamorphosis, evolution and the origins of life (Wald 1958,1963,

GEORGE WALD 11

1964a). As he says in his Nobel lecture: ‘Molecules haven’t taken meout of bioIogy. They’ve drawn me more deeply into it’ (Wald 1968).

So much for rods, but what about cone vision? The visual sensitivity of cone vision lies at even longer wavelengths than the porphyropsin system. Do cones have a third vitamin A that absorbs at yet longer wavelengths? Since chicken retinas have mostly cones, George promptly got chicken heads from a kosher butcher, dissected the retinas and extracted their photosensitive pigments. By exposing the solution to deep red light, he was able to identify a photosensitive pigment with its absorption maximum close to the maximum sensitivity of human cone vision and of daylight vision of chicks (Wald 1937). He called it iodopsin, but since it was mixed with rhodopsin, he could not prove that it bleaches to retinene and vitamin A until years later, when it became possible to synthesize visual pigments from retinene and their protein component, opsin (Wald et a1 1955).

This line of experiments was interrupted by World War 11, when George worked under contract to the armed services, measuring the sensitivity of human vision in the ultraviolet and infrared, and the spherical and chromatic aberration of the human lens (Wald 1945, Wald & Griffin 1947, Griffin et a1 1947). But, as soon as the war was over, George went back to molecules, now with a group of new graduate students, myself included, and with Paul Brown, his co-worker until he retired.

The big break came immediately after the war from the laboratory of the organic chemist and spectroscopist R. A. Morton in Liverpool. While on night watch for German aircraft and missiles, Morton had been fighting boredom by looking at absorption spectra. And, studying their spectra, he decided that retinene must be vitamin A aldehyde. As soon as the war was over, heand his colleagues found a way to oxidize vitamin A to the aldehyde and showed that it was, indeed, identical with retinene (Ball et a1 1946).

This meant that we now had an ample source of retinene - now called retinal ~

because we could readily buy vitamin A, either as a concentrate from fish liver oils or as synthetic crystals. It also led us to isolate an enzyme system from retinas which catalysed the interconversion of retinal and vitamin A (Wald 1950a).

Shortly, Paul Brown found that one could regenerate rhodopsin simply by mixing retinal with bleached rhodopsin, or with its protein part, opsin, in the dark. No enzymes needed, no energy source - a spontaneous, energy-yielding reaction (Wald & Brown 1950). That suggested that we should be able to start with vitamin A and synthesize rhodopsin by adding the retinal reductase system and opsin in the dark. And that worked, too (Wald & Hubbard 1950, Hubbard & Wald 1951). But now we ran into a puzzle: when we used a concentrate of fish liver vitamin A, we got rhodopsin, though with its absorption spectrum shifted to somewhat shorter wavelengths, but when we used synthetic, crystalline vitamin A, nothing happened.

12 HUBBARD & WALD

Some years earlier, the Hungarian organic chemist Leonor Zechmeister (1944) had shown that carotenoids can assume different shapes by forming cis or trans isomers around their various double bonds and that light promotes these isomerizations, if you add a trace of iodine. And indeed, illuminating solutions of our vitamin A crystals in the presence of iodine made it able to form rhodopsin. What’s more, we found that, with retinal, you didn’t need iodine. You got even better yields of rhodopsin if you just irradiated retinal by itself (Hubbard & Wald 1952).

Working with a number of organic chemists, we shortly were able to show that only a single, bent and twisted, sterically hindered shape of retinal - the 11 -cis isomer-combined with opsin to form rhodopsin. Another bent shape, the 9-cis isomer, also formed a photosensitive pigment with opsin, but with a slightly different absorption maximum. (This was the pigment we had gotten from the fish liver oil vitamin A.) None of the other isomers did anything (Oroshnik et a1 1956).

We had shown in 1952 that the retinal that comes off when you bleach rhodopsin is all-trans, so that a cycle of stereoisomerizations between 1 1-cis- and all-trans- retinal is part of the bleaching and synthesis of the visual pigments. But it took until 1958 to realize that what light, in fact, does in vision is to stereoisomerize the chromophore of visual pigments (Hubbard & Kropf 1958). We didn’t really get to understand that until after we had analysed the rhodopsin system of the squid and begun to work with vertebrate rhodopsins at low temperatures (Hubbard & St. George 1958, Hubbard et a1 1959). Here, Professor Yoshizawa made a major contribution by showing that, if you illuminate rhodopsin at liquid nitrogen temperatures, the product is a more intense pigment than rhodopsin itself. And that, on warming this pigment in the dark, it is transformed in stages until it finally splits into all-trans-retinal and opsin (Yoshizawa & Wald 1963).

Like many vision workers, George was always intrigued by human colour vision. In the 1960s, he therefore developed psychophysical methods to determine the spectral sensitivities of the blue, green, and red receptors of colour-normal and colour-blind subjects. In parallel, Paul Brown measured the absorption spectra, first, of tiny patches of the cone-rich fovea of monkey and human retinas and, eventually, of single foveal cones. Putting the results of both types of experiments together, they determined the absorption spectra of the three photosensitive pigments of human colour vision and showed that all three have an 1 1-cis-retinaldehyde chromophore and must therefore contain different opsins (Wald 1964b, Brown & Wald 1963).

Exciting as all this was, it did not touch the question central to everyone interested in vision: what about visual excitation?

Hecht and his colleagues had shown already in 1941 that the absorption of a single quantum is enough to stimulate a dark-adapted rod (Hecht et a1 1942). How can isomerizing just one rhodopsin chromophore possibly do that?

GEORGE WALD 13

FIG. 2. Circa 1967.

As early as 1950, George suggested that maybe bleaching turns rhodopsin into an enzyme which then amplifies that event (Wald 1950b). In 1965, he published a note in Science, suggesting a way to achieve even greater amplification. Reasoning by analogy with the cascade of reactions during blood clotting, he suggested that bleaching rhodopsin might trigger a cascade of enzyme reactions (Wald 1965). And that is close to the way it has turned out to be.

That is the vision story. I have left out many colleagues in other laboratories with whom George collaborated and consulted. I have also left out the undergraduates, graduate students, and postdocs who made George’s lab the exciting place it was. And not only the work, but our daily lunches at which we discussed politics,

14 HUBBARD & WALD

literature, art, occasionally even science - anything that seemed interesting. Nor have I mentioned other molecular and physiological processes that George and his students explored over the years.

I’ve also left out George’s teaching. His biochemistry course and, later, his famous introductory biology course, Natural Sciences 5: The Nature of Living Things, where together with a succession of young colleagues, he introduced thousands of undergraduates to cosmology, to atoms and molecules, and to organisms. George loved to teach. And he took his love of teaching and of biology and life into the political activities and the other things that occupied him in his later years.

111.

When George started Nat. Sci. 5, as it was called, he was doing a very rare thing for someone who had already established a reasonably strong reputation in his field: volunteering to teach the freshman introductory course in his subject. This was terrifically important to him. He felt that the most important thing he could do as a teacher was to reach young people before they had been turned-off the subject, and get them excited by it. It is worth mentioning in this context that, in his last years, the doctor who took care of him (and did a wonderful job of it), was a man who had entered Harvard as a Sanskrit scholar and had just taken Nat. Sci. 5 on a whim, as his one science course, then became so fascinated that he went on into biology and to medical school and became a doctor.

George’s involvement with his students was very much a two-way process. As he was teaching them, he was also learning from them, and the lessons extended beyond biology. In December 1967, when he received the Nobel Prize for Physiology or Medicine, at the US Embassy’s dinner for the American Nobel Laureates, he did the extremely unusual thing of, rather than saying the standard, formal things that one does on these occasions, making a statement of opposition to what the USA was doing in Vietnam. This was considered by many people to be a highly insulting thing for him to do in this forum, but he felt he could do no less. Two years earlier, he had been among the signatories of the first open letter to the New York Times protesting against the war in Vietnam. He had always been interested in politics, but the Vietnam War really brought it home because his students were being drafted, and were coming to him for advice, with questions about what they should do.

The Nobel Prize provided George with a platform from which he could focus a spotlight on his political concerns. On March 4, 1969, he took part in a teach-in, held at MIT about the Vietnam War, and there delivered a speech he called, ‘A Generation in Search of a Future’. That speech changed his life. In a way, it was the culmination of a career as a teacher. It was published in full as the entirety of the

GEORGE WALD 15

New Yorker’s ‘Talk of the Town’ section; it was published in full in the Boston Globe and in newspapers around the world; it was translated into over 40 languages, anthologized in books, and released as a phonograph album.

With that, at 63, George started a new life. In a way, it was the next thing to do. He had received the Nobel Prize for his work in biology, and I think there was a bit of a feeling of ‘What more can one do here?’ He did continue to do research, especially in the summers in Woods Hole, but for the next 25 years politics and social action would be his primary concerns.

As he shifted from science to politics, George found himself regularly facing critics who would accuse him of talking about things that were ‘outside his field of expertise.’ But, he profoundly disagreed with the idea that people trained as lawyers, for example, were better qualified than a scientist to talk about the world’s problems. As he always put it, ‘I am a biologist and my field is life.’ Far from feeling that he was venturing outside his field, he felt that he was applying all the knowledge he had absorbed over the years, and that his political work was intimately connected with his scientific work.

The move into politics, though, brought about some profound changes in his life. Soon he was taking pride in new honours, such as being arrested on the steps of the United States Capitol along with Dr Benjamin Spock, author of Baby undCbild Cure. He continued to be active in opposition to the War, travelling to Vietnam and all over the world in an attempt to end the killing. When the War ended, his main foci became antinuclear activism and international human rights. He came regularly to Japan for the activities around Hiroshima Day, as well as travelling to what, from the American government’s point of view, were unpopular places, such as China and Nicaragua, as well as to Iran during the hostage crisis, always trying to initiate dialogue rather than armed confrontation. He also took part in various international tribunals, assembling testimony and documentation on dictatorships around the world, and met with heads of state in the effort to promote a better world.

George taught his last class in 1977, and his students gave him a conga drum as a going-away present, which he proceeded to play for them while a local storyteller, Brother Blue, performed. He was 70, the obligatory age of retirement and, by that time, had also rather lost his taste for teaching that class. At times he would say that he thought any great class should only last about 10 years. Students already knew all the jokes before he got a chance to deliver them, and he also was bothered by the fact that, after the excitement of 1960s and the way the students had been so interested in the world around them, now he was getting a generation of students who were strictly interested in what their grades were going to be. That just wasn’t enough for him. For example, there had been a period where, when it came time to do their animal dissections, students had protested that they did not want to kill the animals. He had held a special meeting where they could discuss this situation, and

16 HUBBARD & WALD

FIG. 3. Last Nat. Sci. 5 lecture, May 1977.

he did that several years running. When the year came when no student objected to doing animal dissections, that bothered him profoundly.

It is important that one not over-stress the change in George’s life from being a scientist and teacher into doing politics, and then in later years when he began to ask other sorts of questions. In a speech called, ‘The Origin of Life’ which he began to deliver around 1960 he said, ‘When I was a young student I used to be told that a scientist always asks how, but never why. I have come to think that a degraded view of the scientist. To be sure we ask how again and again, but if we have had the good fortune to be answered there comes a time to ask why.’ More and more in his later years that became a question that fascinated him.

George was, in his own way, a profoundly religious man. That was one of the things he loved about Japan. He would, when visiting, stay in an inn in a Zen monastery. His interest in religion was polymorphous; he was not interested in dogma in any way. He was trying, as in science, to understand how the world worked. Over the years, he had developed a number of popular science lectures. ‘The Origin of Life’ was followed by a speech called ‘The Origin of Death’. The last such speech, which he deliveredfor many years in various changing forms, was called ‘Life and Mind in the Universe.’ It would start off with some physics and cosmology, exploring questions like how it comes to be that everything but water gets denser as it

GEORGE WALD 17

freezes, and only water expands and therefore floats on itself, without which life on Earth would be impossible. He would wonder how that happened, and it would be interesting watchng him deliver this speech, because to non-scientists it was utterly fascinating, while scientists would divide between some being fascinated and some feeling that he was stepping over too much into religion.

He would go on to talk about ‘consciousness,’ which to him had become one of the most fascinating of subjects. He had done all these studies of frogs, and frogs’ eyes, and how all of that process worked, and he began thinking about the fact that he still had no idea whether a frog could ‘see’ in the sense that he understood himself to be able to see, and that all the experiments he could design could only demonstrate that a frog could react to stimuli and could never answer that question. He went on to muse about something that he referred to as ‘universal mind’. This did not exactly mean a god or gods, it meant an organizing principle for the universe or some reason why things worked. Such questions had always interested him in private, and when he began to be less directly involved in politics they took up more and more of his attention. He never claimed to have answers, but was constantly trying to get a deeper understanding of the world around him.

I want to end by giving one last quotation because, towards the end of his life, I taped George’s memoirs. This was at a period when he had not done any active science for many years, but, at the end of the taping, the thing that he wanted to do was talk about science. To him, that was the discipline that he had used in everything he did throughout his life and he never ever felt that he had left it. He felt, therefore, that it would be appropriate to end his memoirs with something he had often told his students:

‘One of the most important sources of human happiness is to find an unachievable objective. That sounds strange, but, in this life, there are many things you want. You want to find someone you can love, you want to build a home, you want to have children. And these things you will do, but somehow the finding will never quite come up to the dreams that went into it. So it’s important to find one goal that never stops being a goal, where you can have little victories, but they are just incidents in that bigger thing. Science fulfils such a role, and I think that is an exceedingly important part of becoming a scientist. One becomes ever so deeply involved with the realization that every answer you find just raises new questions, and one need never fear that one will come to the end of the enterprise.’

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DISCUSSION

Dowling: I was pleased, Ruth and Elijah, that you emphasized George’s teaching, because this was what drew me into science and into the study of vision. Indeed, I