Gobind’s last graduate student

Abstract

Written on the occasion of his 100th birthday, this is a personal account of my time as a graduate student with Nobel laureate, H. Gobind Khorana, at the Massachusetts Institute of Technology from 1996 to 2000.

How it all began (for me)

I first “met” Gobind through one of his papers about bacteriorhodopsin. It was the year 1995. Yes, at the time of writing it is the year 2022 and I am 50 years old, or as one of my undergraduate students once put it, half a century. She (innocently?) hailed this truth at me with her question “Dr. Klein, what does it feel like to be a half a century old?.” We are also celebrating Gobind’s 100th birthday this year. Figure 1 shows a birthday note that Gobind wrote to me while I was a graduate student (Fig. 1). He did not miss the opportunity to comment on the state of mess of my desk (birthday resolve).

Fig. 1.

Birthday note. (Photograph used with permission of the owner, Judith Klein-Seetharaman)

I first stumbled across his name when I was an undergraduate student in Germany where I was studying two full Diplom degree courses, one in Chemistry and one in Biology at the University of Cologne. Probably, this was the first indication of my lifelong love of interdisciplinarity — that I could not make up my mind if I should study Chemistry or Biology, so I studied both. I only knew one other student at the University of Cologne, who studied two full degree courses, one in Chemistry and one in Medicine. One afternoon, I sat in the Cologne Chemistry Department library, the sun shining through the glass windows of the 1970s concrete building, looking at printed journal collections displayed prominently, at a time when nothing was available online except role playing games through simple command shells. I had just returned to Cologne from a year abroad, spent in the laboratory of Professor Jim Barber at Imperial College of Science, Technology and Medicine in London, where I completed my diploma thesis on cytochrome b559, a protein that is part of the photosynthetic reaction center, PSII. It is a heme-containing protein with redox activity that paradoxically displayed two redox potentials. At the time, the paradoxical behavior was explained with the comment “it’s a conformational change.” It was treated as a black box, and left me wondering what is a conformational change. When I accidentally came across Gobind’s article about conformational changes in bacteriorhodopsin upon light activation, I admired the great detail at which he described the path of proton transfer through the protein across the membrane. I wrote to him and said that I would like to do my PhD with him. It is important to realize that the culture of doing science in Germany was very different from that in the USA. In general, regardless of subject, there were no graduate programs in Germany at that time. You just wrote to a Professor and agreed to work together. So, it came as a surprise to me when Gobind replied (freely cited from my memory): “That’s great, but first you need to apply and get admitted to MIT. You can choose if you want to apply to the biology or the chemistry graduate program. But before you apply, I’d like to meet you first.”

Gobind was planning a trip to England with his wife Esther and he bought a plane ticket for me to meet them in England. They took me to an outdoor pub in the British countryside. I was very nervous about what was clearly setup to be an interview, but Gobind and Esther were simply lovely to me and all I remember from the meeting is that they were so nice to me and that I had a sandwich with cheddar cheese and pickles. So, I applied to the PhD program in chemistry at the Massachusetts Institute of Technology (MIT) with the statement that I want to work with Gobind. As a backup, I also applied and was admitted to the PhD program at Yale University. There, I had identified a German scientist, Professor Dieter Söll through the alumni list of the Studienstiftung des deutschen Volkes which had supported my undergraduate studies in Germany. So, I bought my own plane ticket this time to come and visit both MIT and Yale to make the decision where to carry out my PhD. I met with the postdocs in Gobind’s lab at the time who openly advised me that he was a slave driver. This did not scare me. After all, I had competed two full degree courses in the time of one, so hard work, sleepless nights of studying and worried looks from my mother telling me to please take it easy was something I was used to. At Yale, the graduate students told me that New Haven is so unsafe that you cannot do experiments after 8 pm, and a student of the lab had just been attacked by a thief after hours. Furthermore, the work in the lab at Yale was very nucleic acid focused, and my interest was in proteins. I decided to work with Gobind/MIT. Many years later, at one of the Gobind alumni meetings that used to take place every 4 years, I found out that Dieter Söll was a former postdoc of Gobind. I learned that Gobind himself had started out with nucleic acid chemistry and transitioned to proteins after he had solved the most fundamentally important of all nucleic acid questions — which amino acids are encoded by which nucleotide combinations, a discovery for which he was awarded with the 1968 Nobel Prize in Physiology and Medicine. As a naïve and ignorant undergraduate student in Germany (not sure that my young age at the time is a valid excuse), I had no idea that Gobind was a Nobel Laureate when I contacted him, and the decision to work for him was purely based on his work on conformational changes in proteins (and no mugging threats after hours around building 68, the biology building, at MIT).

Thus, in 1996, I joined the lab of Har Gobind Khorana at the Massachusetts Institute of Technology, as his final graduate student. The next to final graduate student was Kewen Cai, who had started and thus was destined to graduate (and did graduate) one year before me.

A family away from home

Gobind and Esther became my second parents. Separated from my mother by an ocean and a 7-h time difference, they took care of me as if I was a family member. Gobind met me with a taxi at the airport (see welcome letter in Fig. 2A) and helped me carry the stereo system that I had brought from Germany in addition to my suitcase into the MIT dormitory. Esther drove me to the MIT student exchange where you could buy furniture left by graduating MIT students. My first night at the dorm was a bit scary — the dorm room had iron bars and Vassar street had a drug rehabilitation facility and a recycling center next to it. Maybe MIT wasn’t safer than Yale after all. I called my mother who bought a plane ticket and came to visit me on the next possible flight. She deemed the arrangement safe and so I began my PhD studies at MIT. Gobind and Esther made it a special point to meet my mother (Fig. 2B,C). Gobind and Esther organized regular events such as the annual apple picking near their summer house in New Hampshire. They were an integral part of all major personal events of lab members such as weddings and baby showers. Esther and Gobind were one person to us, as far as I could tell he was not functional without her. Esther did everything for Gobind. For example, she drove him everywhere he needed to go. Julia was also a frequent visitor of the lab, as she prepared all publication-ready graphics for our papers (see more on this below). Last but not least, Judy Carlin, Gobind’s personal secretary, was his official face, and only lab members were allowed to contact him directly, through the back door to his office only accessible from the lab computer room. People who irritated Gobind were all buffered by Judy. There were two types of people that were in this category. One, people from India who were proud of Gobind’s achievements. Gobind was born in what was then India but is now Pakistan, and he went to Manchester, England, to do his PhD, which he completed in record time. His fellowship required him to return to India, and when he went there, he couldn’t find a job. This made him very upset and he left India to continue his career in North America. The second type of people were those seeking nominations for the Nobel prize. There are many scientists who have not received the award themselves, and aim to benefit in other ways from those people who did receive the award.

Fig. 2.

Gobind and Esther: a family away from home. A Fax sent to me in Germany planning my arrival at MIT. B Note on letter head. C Note regarding my mother’s visit at MIT. (Photographs used with permission of the owner, Judith Klein-Seetharaman)

A picture of the lab at the time I was a graduate student

I have not taken many pictures and the few that I had have been lost in the many household moves of my life, except two. One is a picture of the Khorana lab at the time in front of a lab poster (Fig. 3A), alongside the planning of the same poster (Fig. 3B). The second is a group picture of the combined RajBhandary and Khorana labs from July 1997 (Fig. 3C). My memory is also terrible, so this article may not be fully accurate. I am also not very entertaining, so please excuse the “stream of consciousness” nature of this article. Given the opportunity, I gladly tell stories about Gobind, so some of them have made it through the years saved in various corners of my brain. I will aim to put them here on paper, digital of course. And I am a hoarder by nature, so I have kept some of the hand-written notes Gobind has left for me — on paper in the widest possible sense including lab tissue and memos typed up by Judy Carlin, his magical administrative assistant (next to Gobind in Fig. 3C).

Fig. 3.

The Khorana lab at the time I was in his lab. A The Khorana lab sometime after I joined in 1995 in front of our lab poster. Top row, from left to right: E. Getmanova, J. Klein, Y. Itoh, H.G. Khorana, P.J. Reeves, J. Kim, L. Niu. Bottom row, from left to right: J. Hwa, K. Cai, M. Lowen. B The lab poster from A during its preparation phase. Outline and note from Gobind. C The annual group picture of the combined Khorana and RajBhandary labs in July 1997. Front row: P. Reeves, A. McInnis, U.L. RajBhandary, A. Kowal, K. Cai, H.G. Khorana, J. Carlin, U. Varshney, H.J. Drabkin, Y. Li. Back row: R. Vaidyanathan, K. Cha, C. Bruel, J. Klein, H-J. Park, S. Gite, J. Kim, J. Hwa. (Photographs used with permission of the owner, Judith Klein-Seetharaman)

Gobind’s reputation as a slave driver

The notion propagated by Gobind’s postdocs of him being a slave driver had been juiced up with the following story. I have no idea if it is true or a myth, but for all I know it could have been true, because Gobind was definitely obsessed with being worried about that people weren’t working hard. True or not, here is the story. Apparently, Gobind’s first postdoc was also German. He arrived in the lab and had his first meeting with Gobind in his office. There, he asked Gobind about work-life balance and what his expected working hours were. The next day, he found a plane ticket back to Germany on his lab bench.

This story might not be true, but the following stories definitely are since I experienced them firsthand, and they do paint a consistent picture. And which Professor doesn’t want their students and postdocs to work hard?

First, there was the seminar schedule. There were three seminars per week, strategically placed as follows: one on Monday morning at 8 am, one on Wednesday at 12 noon and one on Friday at 3 pm, that used to run until about 4:30–5 pm. Obviously, the timings of these seminars were placed to ensure that people were at the lab from at least 8 am until 5 pm every day from Monday to Friday. The Monday seminar was shared with Prof. Tom RajBhandary, the PI of the neighboring lab. Each week, two talks were scheduled, one group seminar presentation from Tom’s lab and one from Gobind’s lab. These seminars were the most serious presentations because you had to present in front of everyone. Especially Gobind’s approval was feared because he would ask harsh questions and judge your presentation very rigorously. You could also never present something that he didn’t already know you were working on. If you conducted an experiment without telling him and presented the result on Monday, he used to be furious. We had to prepare a typed seminar report for each presentation and leave a printed copy on each lab members desk (including Tom’s) the Friday afternoon before the seminar presentation on Monday morning. The person whose seminar it was on Monday had priority on all the instrumentation as this was the most productive time in generating research results. Because we had to write down our work in paper style format, and thus reflect on our own work and how to best interpret and present it, it would become obvious what kind of experiments we should have done. It often becomes clear only through reflection what should have been included as a positive or negative control, or that an experimental output should have been recorded for longer times or at more frequent intervals or different concentration ranges.

Gobind definitely wished people would work on weekends, although he didn’t force people to do so. Gobind himself came to the lab on Saturday mornings, in itself a reason to come to the lab on Saturday because you could meet with him informally without making an appointment. While coming to the lab on Saturday was entirely voluntary, it was rewarded by Gobind — with a donut. Gobind would bring in a big box with a dozen donuts, the type where every single donut was different. Each person in the lab would have their favorite donut and eat only “theirs.” After a while, we figured out that Gobind had memorized which member of the lab likes which donut (Fig. 4). One Saturday, while I was working in the lab, I received a phone call from one of the postdocs who asked me “Hey Judith, can you eat the Boston crème for me?”.

Fig. 4.

A box of donuts appeared every Saturday in the computer room at the lab. Shown is here how I think a classical donut box looked like in Gobind’s brain. More pictures of the people depicted here are also shown in Fig. 3A, C

In all the time that I was a graduate student (from February 1996 to September 2000), I took exactly one holiday. I left on Friday and came back Sunday night, in time for the Monday morning presentation. I went to the Grand Canyon, missing exactly one Friday afternoon seminar and a donut on Saturday. Gobind grieved me for this trip for the remainder of my PhD: “if only you hadn’t gone on vacation, this question would long be solved.” This was about an experiment in which I measured peptides released from rhodopsin to indicate accessibility around the conserved disulfide bond using paper chromatography. This experiment was never published, possibly my punishment for taking vacation. It was actually that trip that inspired the topic for my comprehensive exam, which was supposed to be on a topic unrelated to the PhD thesis topic. Grand Canyon — which incidentally I thought was the most beautiful place on earth — is home to a type of cactus that is purple. I always had a soft spot for plants (the first document I ever wrote on a computer was a report on evolution of plants that I spent months on as a high school student — an interest clearly inspired by my mother who worked on plant anatomy at the time and created beautiful pencil drawings of stomata microstructures). Gobind really didn’t need to worry about my work ethics: I wrote and defended my comprehensive exam on the chemical nature and biological purpose of what makes these cacti in the Grand Canyon purple. Ironically, I now live near the Grand Canyon, in Phoenix, Arizona, about 3.5 h drive from it. It is still the most beautiful place on earth to me. And I work in a chemistry department (the School of Molecular Sciences at Arizona State University) that is home to world class research in the biophysics and structural biology of photosynthesis. The purple-colored cactus still grows on the plane in the center of the Grand Canyon and actually as I now know — everywhere in Arizona.

The happiest I ever saw Gobind was when he found a yellow sticky note from the other graduate student at the time, Kewen Cai, on my desk. She had left a note for me to take out her samples from the heat block at midnight. This was obviously independent evidence that both Kewen and I were working late at night and Gobind never doubted my work ethics again.

The yellow sticky notes

Since email wasn’t really much of a thing yet when I was a graduate student, the main way to communicate with Gobind was in person during the daytime, via fax when he was traveling or via sticky notes or really any writable surface including paper napkins anytime else (Figs. 5 and 6). Gobind never stopped thinking about science. He would bring yellow sticky notes to the lab that he had written at home in the night when he woke up, labeled with the time, e.g., 3 am, when he was thinking to remind me of something or if he had a scientific idea or thought of an interpretation of a result. Unfortunately, I also have many sticky notes commenting on the terrible mess on my desk and my lab bench (Fig. 6).

Fig. 5.

Primary means of communication: in-person meetings and notes. Shown here are some of Gobind’s hand-written notes or typed up notes via Judy Carlin. A Regular note. B Yellow sticky note. C Paper napkin note. D Typed up note. (Photographs used with permission of the owner, Judith Klein-Seetharaman)

Fig. 6.

More notes. A Small paper note. B Paper napkin, taped to my lab bench with purple tape found on my bench. (Photographs used with permission of the owner, Judith Klein-Seetharaman)

Every year in August, Gobind would spend the entire month with his collaborators in Japan. He loved going there and even though you might expect that “when the cat’s away, the mice can play,” it was one of the most productive times of the year. Due to the time difference, we were double as efficient than usual because Gobind would send us instructions that arrived by fax during the night, so were waiting for us when we arrived at the lab in the morning, and gave us enough time to do experiments or write papers during the day and send the results of the day’s work by fax to Japan in the evening US time. He would receive our faxes when he started work in Japan in the morning, just in time to give us feedback while we were sleeping. His faxes would then arrive just in time for our arrival at the lab the next day. A sequence of such fax exchanges is shown in Fig. 7. They highlight a lot about Gobind — his sense of humor, references to Julia and paper writing (see more details below), experiments, collaborators, presentation preparation and more.

Fig. 7.

Faxes from Japan. A Fax from Japan on 10/16/1998. B Slide referenced in the joke from A. C Fax from Japan on 10/19/1998. D Fax from Japan on 10/21/1998. (Photographs used with permission of the owner, Judith Klein-Seetharaman)

How we published papers

The examples from the faxes in Japan are not exaggerated. Gobind was very particular about anything that left the lab, especially papers and posters. He worked very meticulously with us to perfect those and nothing would be published without his detailed involvement and approval. This process is illustrated in part in Fig. 8. The process obviously starts with the accomplished experiments (Fig. 8A). Next is the outline (in hand writing, on paper, Fig. 8B). Then, multiple rounds of writing and re-writing (Fig. 8C). The perfectionism in this last step sometimes could go to extremes and while I might have been disgruntled by it occasionally at the time, I am guilty of it myself with my own students and postdocs, and I will be eternally grateful for the training in science writing and presentation I received from Gobind. This is particularly important considering that I am not a native English speaker. Every publication went through numerous rounds of rewriting, see Fig. 8C for an example. Obviously, the intention was to improve the presentation of the paper, but oftentimes the rewritten version would be worse than the previous version so it had to go through another round of rewriting. This iterative process would result in papers that were “written in stone” — every single word was carefully evaluated for its legitimacy to appear at the specific position in the paper. For Gobind, the rigor and accuracy of what was written in our papers was of paramount importance. He used to say that while interpretations would come and go, the experiments themselves have to stand the test of time. Anyone should be able to obtain all of the information needed from a publication to repeat the experiment and get the same result, even if in 20 years with additional knowledge we might interpret these experiments differently.

Fig. 8.

The paper writing process. A Step 1: a note. B Step 2: an outline. C Steps 3-n: multiple rounds of revisions. (Photographs used with permission of the owner, Judith Klein-Seetharaman)

One word is all that was left of the introduction page of my first printed paper (unfortunately, I didn’t keep a copy — I was probably too ashamed). It is the year 1999 and we did have a common computer room in the lab. Word processing and graphing were the only programs available at the time. Even though we created the first draft of our papers using a computer, including images, Gobind used yellow legal paper with lines (see Fig. 8B) to hand-write the bulk of the text, and his daughter Julia, a graphic designer, hand drew the images for us. Julia creating the final images served the function of having a uniform style with consistent font styles and sizes across all of his papers regardless of who in the group had generated the data. For the text, Gobind would cut out words from the printed paper draft and glue them onto the yellow paper, where he hand-wrote the remainder of the text. The amount of text cut out varied and increased with the version number of the draft. When getting to a near-final version, we were no longer allowed to create versions of the paper electronically. His secretary, Judy Carlin, would be the only one allowed to type up the manuscript at that stage and make any changes to it. This was his way of version control.

The high quality of the outputs of this rigorous publication writing process resulted in enormous pride in the result. Gobind would establish a logical sequence to all of his work by placing a footnote associated with the title of the paper. “This is paper number 2 in the series “Structure and function of rhodopsin,” the previous paper in the series is reference [1]”. I tried many times in my career to create such a series but my level of focus never allowed me to continue beyond paper #1 in a series. Gobind’s productivity was so incredible, for example, he published one entire issue of the Journal of Biological Chemistry (JBC), in which every single paper was from his lab. His memory was also incredible. He would be able to cite from these papers, knowing the precise page number on which to find crucial information published in these papers. He would take the printed book (a very fat issue of JBC!), open the page in question, and point at the sentence, and let out a little giggle and smile, satisfied at the notion that he was right. The key papers from his career can be found collated in a book, where he wrote introductions to the different phases of topics studied in his career (Khorana 2000).

The balance between focus and change in research

Gobind’s philosophy was to change the research area he was working on every 10 years. The central dogma of biology delineates the process by which DNA is transcribed to RNA and translated to proteins, and while we now know that there are a lot of deviations from the central dogma, it is still essentially true, and Gobind made crucial contributions at the logical progression along this dogma. First, he worked on DNA, and deciphered the genetic code through chemical synthesis of polynucleotides. Then he worked on RNA, creating the first synthetic gene for a tRNA, along the process inventing the polymerase chain reaction or PCR. The tRNA gene paved the way to many other synthetic genes, including the opsin gene which was the basis for my own work with him. I made over 150 point mutations in opsin as a PhD student and the process was enabled by the facile replacement of fragments with defined nuclease recognition sites, allowing cassette mutagenesis. It was only logical for him that the next frontier were proteins, and he decided to focus on those proteins that are most challenging to study, the membrane proteins. At that 10-year break point when he decided, membrane proteins were next, he gave each of his postdocs one membrane protein to work on. At the time, his lab covered the entire 7th floor of building 56, the chemistry building at MIT. There were probably some 50 postdocs working for him at the time (this is a wild guess). Bacteriorhodopsin behaved best and became the first model protein for membrane protein research. You could do anything you want with bacteriorhodopsin, including fully denaturing it and refolding it in vitro, still the only helical membrane proteins you can do this to today. This means that Gobind founded the field of membrane protein research. After 10 years, he left bacteriorhodopsin and moved to mammalian rhodopsin. Rhodopsin was the first G protein coupled receptor (GPCR) studied in vitro, paving the way to the study of GPCR’s. Thus, he pioneered not only what he is famous for and what he received the Nobel prize for — the genetic code — but also the fields of membrane proteins and of GPCRs. GPCRs are the pharmacologically most important protein family with more than 50% of all drugs on the current market targeted at GPCRs. Thus, the importance of his work on rhodopsin cannot be understated for human health and drug discovery.

Legacy as an interdisciplinary scientist

Gobind is a chemist by training, yet he put the chemistry to work to make several of the most remarkable discoveries in biology. When I joined Gobind’s lab, I came educated as a chemist and as a biologist, yet most of my work was in biophysics. Instead of sending the samples of purified rhodopsin off to our collaborator Wayne Hubbell at UCLA, I wanted to study them myself. A bit like a spoilt child, I used to say “I want to do that thing” — refusing to let someone else do what I considered the fun part of research — spectroscopy — with my samples that I spent a lot of time and effort preparing. Since that wasn’t easily done with a collaborator at the other side of the continent, I went to the basement of the chemistry department at MIT instead and asked the NMR facilities manager to teach me how to use NMR instruments. I also took classes in NMR spectroscopy theory. As a result, I was able to collect the very first ever NMR spectrum of a GPCR, rhodopsin. This opened the door to many new types of experiments, and Gobind immediately put several postdocs onto my projects. While I was originally disgruntled about this, I benefited greatly as we published many collaborative papers together (Table 1) and I saw many of my ideas reach reality more rapidly than would have been possible alone. I became the biophysics representative of the lab. Gobind would send me to the biophysical society meeting every year to represent our lab and to find out what other biophysicists were working on. I sometimes wished I had studied physics in addition to chemistry and biology. Then, of course, once I moved after my PhD to Carnegie Mellon University to work with Raj Reddy, Jaime Carbonell, Roni Rosenfeld, and Yiming Yang in the School of Computer Science, I wished I had studied computer science. Having already jumped across the boundary between chemistry and biology with a PhD in biological chemistry, I managed to communicate with the computer scientists who had never worked on proteins by using analogies between words in English and chemical groups of amino acids as words in protein sequence language, in the NSF-funded biological language modeling project (Klein-Seetharaman and Reddy 2002; Ganapathiraju et al. 2003, 2005). Thank you Gobind for nurturing me in collaboration and cross-disciplinary training.

Table 1.

Publications arising from my PhD with H. Gobind Khorana

1	Hwa, J., Reeves, P.J., Klein-Seetharaman, J., Davidson, F. and Khorana, H.G. (1999) Structure and Function in Rhodopsin: Further Elucidation of the Role of the Intradiscal Cysteines, Cys-110, -185, and -187, in Rhodopsin Folding and Function. Proc. Natl. Acad. Sci. USA 96, 1932–1935
2	Cai, K., Klein-Seetharaman, J., Farrens, D., Zhang, C., Altenbach, C., Hubbell, W.L. and Khorana, H.G. (1999) Single Cysteine Substitution Mutants at Amino Acid Positions 306–312 in Rhodopsin, the Sequence between the Cytoplasmic End of Helix VII and the Palmitoylation Sites: Sulfhydryl Reactivity and Transducin Activation Reveal a Tertiary Structure. Biochemistry 38, 7925–7930
3	Klein-Seetharaman, J., Hwa, J., Cai, K., Altenbach, C., Hubbell, W.L. and Khorana, H.G. (1999) Single Cysteine Substitution Mutants at Amino Acid Positions 55–75, the Sequence Connecting the Cytoplasmic Ends of Helix I and II in Rhodopsin: Reactivity of the Sulfhydryl Groups and their Derivatives Identifies a Tertiary Structure that Changes Upon Light-Activation. Biochemistry 38, 7938–7944
4	Altenbach, C., Klein-Seetharaman, J., Hwa, J., Khorana, H.G. and Hubbell, W.L. (1999) Structural Features and Light-Dependent Changes in the Sequence 59–75 Connecting Helices I and II in Rhodopsin: A Site-Directed Spin Labeling Study. Biochemistry 38, 7945–7949
5	Cai, K., Klein-Seetharaman, J., Hwa, J., Hubbell, W.L. and Khorana, H.G. (1999) Structure and Function in Rhodopsin. Effects of Disulfide Cross-Links in the Cytoplasmic Face of Rhodopsin on Transducin Activation and Phosphorylation by Rhodopsin Kinase. Biochemistry 38, 12,893–12,898
6	Klein-Seetharaman, J., Hwa, J., Cai, K., Altenbach, C., Hubbell, W.L. and Khorana, H.G. (2001) Probing the Dark State Tertiary Structure in the Cytoplasmic Domain of Rhodopsin: Proximities Between Amino Acids Deduced from Spontaneous Disulfide Bond Formation between Cys316 and Engineered Cysteines in Cytoplasmic Loop 1. Biochemistry 40, 12,472–12,478
7	Cai, K., Klein-Seetharaman, J., Altenbach, C., Hubbell, W.L. and Khorana, H.G. (2001) Probing the Dark State Tertiary Structure in the Cytoplasmic Domain of Rhodopsin: Proximities between Amino Acids Deduced from Spontaneous Disulfide Bond Formation between Cysteine Pairs Engineered in Cytoplasmic Loops 1, 3 and 4. Biochemistry 40, 12,479–12,485
8	Altenbach, C., Klein-Seetharaman, J., Cai, K., Khorana, H.G. and Hubbell, W.L. (2001) Structure and Function in Rhodopsin: Mapping Light-Dependent Changes in Distance between Residue 316 in Helix 8 and Residues in the Sequence 60–75, Covering the Cytoplasmic End of Helices TM1 and TM2 and their Connection Loop CL1. Biochemistry 40, 15,493–15,500
9	Altenbach, C., Cai, K., Klein-Seetharaman, J., Khorana, H.G. and Hubbell, W.L. (2001) Structure and Function in Rhodopsin: Mapping Light-Dependent Changes in Distance between Residue 65 in Helix TM1 and Residues in the Sequence 306–319 at the Cytoplasmic End of Helix TM7 and Helix H8. Biochemistry 40, 15,483–15,492
10	Hwa, J., Klein-Seetharaman, J. and Khorana, H.G. (2001) Structure and Function in Rhodopsin: Mass Spectrometric Identification of the Abnormal Intradiscal Disulfide Bond in Misfolded Retinitis Pigmentosa Mutants. Proc. Natl. Acad. Sci. 98, 4872–4876
11	Klein-Seetharaman, J., Getmanova, E.V., Loewen, M.C., Reeves, P.J. and Khorana, H.G. (1999) NMR Spectroscopy in Studies of Light-Induced Structural Changes in Mammalian Rhodopsin: Applicability of Solution 19F NMR. Proc. Natl. Acad. Sci. USA 96, 13,744–13,749
12	Reeves, P.J., Klein-Seetharaman, J., Getmanova, E.V., Eilers, M., Loewen, M.C., Smith, S.O. and Khorana, H.G. (1999) Expression and Purification of Rhodopsin and Its Mutants from Stable Mammalian Cell Lines: Application to NMR Studies. Biochem. Soc. Trans. 27, 950–955
13	Loewen, M.C., Klein-Seetharaman, J., Getmanova, E.V., Reeves, P.J., Schwalbe, H. and Khorana, H.G. (2001) Solution 19F Nuclear Overhauser Effects in Structural Studies of the Cytoplasmic Domain of Mammalian Rhodopsin Proc. Natl. Acad. Sci. USA 98, 4888–4892
14	Klein-Seetharaman, J., Reeves, P.J., Loewen, M.C., Getmanova, E.V., Chung, J., Schwalbe, H., Wright, P.E. and Khorana, H.G. (2001) Solution NMR Spectroscopy of a-15N-Lysine Labeled Rhodopsin: The Single Peak Observed in both Conventional and TROSY-type HSQC Spectra Originates from Lys339 in the Carboxyl Terminal Peptide Sequence. Proc. Natl. Acad. Sci. USA 99, 3452–3457
15	Getmanova, E., Patel, A.B., Klein-Seetharaman, J., Loewen, M.C., Reeves, P.J., Friedman, N., Sheves, M., Smith, S.O. and Khorana, H.G. (2004) NMR spectroscopy of phosphorylated wild-type rhodopsin: mobility of the phosphorylated C-terminus of rhodopsin in the dark and upon light-activation. Biochemistry 43, 1126–1133
16	Klein-Seetharaman, J., Yanamala, N.V.K., Javeed, F., Reeves, P.J., Getmanova, E.V., Loewen, M.C., Schwalbe, H. and Khorana, H.G. (2004) Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR. Proc. Natl. Acad. Sci. 101, 3409–3413

Additional stories

Below are some additional memories that come to my mind.

The dark room

When I first joined the lab, I found my desk drawer to be full of red marker pens. I thought it was amazing that I inherited so many markers, since usually — like pens — markers/sharpies usually have legs and tend to disappear with people. Happy with my pile of pens, I started labeling my first set of tubes, and took them to the dark room. Rhodopsin is a light-sensitive protein and its absorbance maximum is at 500 nm, or yellow light. To avoid activating rhodopsin by light, we need to use red light, far enough from rhodopsin’s absorbance peak so that it is absorbed minimally. We used to use red photographic lights, with low intensity 15-Watt light bulbs. Apart from these dim lights, no other light source could be present or else the samples would be spoilt. When I carried out my first rhodopsin purification, and eluted fractions into the labeled tubes, I realized why there were so many markers in my drawer. The red color of the pens meant that one could not see them under the red-light conditions in the darkroom, and thus all the sharpies in my drawer were essentially useless.

Nanoscience

Gobind cared about our all-round education and sometimes made suggestions on topics for the Wednesday literature club seminar. Once he came to me excited about an article about carbon nanotubes. At the time, I complained, what is the use in that? I did not want to learn about carbon nanotubes since I saw no connection to rhodopsin. Apparently, there was a time in my life that I was focused. In hindsight, of course, it was the beginning of an explosion of the use of carbon nanotubes in all fields of science and technology and I should have listened to Gobind at the time. I have even published several papers on carbon nanotubes in my career since Gobind (Klein-Seetharaman 2005; Allen et al. 2009; Kagan et al. 2010; Kotchey et al. 2011; Kapralov et al. 2012; Andón et al. 2013).

Asserting my identity

I learned quickly that if I wanted to have some degree of control over my PhD, I needed powerful allies. When it came to choosing my thesis committee, Gobind suggested putting his friends. I felt very brave at the time when I asked him that I would like to chose my own committee members. Reluctantly he agreed, and I asked Joanne Stubbe and Peter Kim to be on my committee. This choice paid off in several ways. First, it allowed me to graduate. I had published numerous papers on rhodopsin misfolding (in collaboration with John Hwa) and conformational changes with cysteine reactivity, disulfide bond formation rates and EPR spectroscopy (the latter in collaboration with Wayne Hubbel’s laboratory) but my major novel intellectual contribution was the first use of NMR spectroscopy for studying rhodopsin structure and conformational changes. In addition to presenting the first ever ¹⁹F NMR spectra of any GPCR, and the first ever isotope incorporation in mammalian cells enabling ¹⁵N NMR of any GPCR using ¹⁵N-α-lysine labeling, I extended the concept to ¹⁵N- α,ε-tryptophan labeling. Rhodopsin has 5 tryptophan residues, so we could expect 10 cross peaks in a ¹H,¹⁵N-HSQC spectrum of rhodopsin, 5 for the side-chains and 5 for the backbone nitrogen atoms of the tryptophan residues. However, while the 5 side chains were clearly visible, there were more than 5 peaks with variable intensity present for the backbone, indicating conformational fluctuations present even in the dark state of rhodopsin. In what I was hoping to be my final thesis committee meeting, Gobind asked “But don’t you need to first assign the peaks in the NMR spectrum of tryptophan labeled rhodopsin?” My committee member Joanne Stubbe simply said “No, she doesn’t.” It was not until 7 years after my PhD and 2 of my own graduate students and collaborators later that we succeeded in the assignment of these NMR peaks (Werner et al. 2007).

Retirement

Gobind had no appreciation for people he thought were not dedicated to research. For example, we had a distinguished Professor (I think he was also German) visit our lab once, and he told us how he was about to retire. Gobind would mutter how this proves that this Professor was not a proper scientist, because if he were he would only think about work and not retirement. He pictured himself working in the lab in his lab coat until the moment he would die. Tragically, his life did not end in this way, and he spent several years in an old people’s home. Indeed, it was not science that was with him until he died, but actually his wife, Esther. I always felt that Gobind died the day his wife Esther passed away. It was as if Gobind and Esther were really one person and one could not exist without the other. Esther was everything in his entire life, from driving him everywhere to being a mother to all of us in the lab. Even though he still breathed for a number of years, his mind started deteriorating from the moment she died. He used to wander off from the old people’s home where the other inmates were playing Bingo and Julia had to search all over Boston to find him.

Scientific intuition

Sometimes, we thought that his hypotheses were more based on a hunch than on real data. However, he drove us hard to then collect as much real data as possible to test the hypothesis. In most cases, he turned out to be right, and it was a mystery to us how. As a result, we admired his scientific insight and deep understanding of how proteins work. For example, he was very stubborn about his hypothesis that there is an aberrant disulfide bond in misfolded rhodopsin. There is a conserved disulfide bond between cysteines at positions 110 and 187, and rhodopsin also has a cysteine at position 185. There was really not much evidence for it, but he insisted that the misfolded rhodopsin has a wrong disulfide bond between 185 and 187. The hypothesis really stems from his chemistry background that dictated that something irreversible such as misfolding is likely due to an aberrant covalent bond. A disulfide bond is exactly that — its breakage requires the presence of a reducing agent. While an oxidizer is always around in the form of oxygen itself, a component of the air we breathe, a reducing agent needs to be provided by the cell or the experimentalist. Needless to say, that Gobind was right, as we have proven experimentally in this paper (Hwa et al. 2001).

Monday lunch/Gobind as a human

Mondays after the group seminar, we would have lunch together in Gobind’s office. This is where we found several milliliters of liquid mercury hidden in a metal box in his office (I eventually disposed of this box with the help of the environmental hazards group at MIT). He also told us about what are all the different kinds of oranges there are such as mandarins. The lunch meetings were over when Gobind stood up from his chair. This was the sign that we can all get up and leave. The same signal was given at the parties in Gobind’s house, e.g., at Christmas — when it was time for us to leave his home, he would get up from his chair. Regardless of the location — in the lab, his office, at his home or even a restaurant — Gobind’s unique intellect would enlighten the conversation. For example, I remember an incident of Gobind’s phenomenal and photographic memory when we visited an Indian restaurant in Boston and he recited the more than 20 ingredients and their quantities for traditional Mulligatawny Soup.

The rate-limiting step

Gobind told me to never be the rate-limiting step. Always do everything you can that is in your power. This could backfire and result in him making a comment about my paper, saying it is useless and that I should work on it more. Over time, I learned to distinguish when such a comment really needed addressing and when it was simply a reflection of Gobind being stressed with something else and not having had time to work on my paper. We also had a rule that we don’t do things that Gobind asks of us, unless he would ask at least twice. This would be our signal that it is actually something he truly cares about us doing.

Lab books/paranoia/legal

We often take this for granted, but lab books are actually legally binding entities that can establish intellectual ownership. There was a time when Gobind was considered for a second Nobel Prize and he had to demonstrate that the crucial experiments were done in his lab before they were done in a competitor’s lab. It was not possible to prove this due to missing entries in lab books. Since then he required everyone in the lab to use lab books that generate carbon copies of the entries. Those carbon copies had to be saved and locked up in a fire-proof metal cabinet and they were not allowed to leave the lab with the scientist who did the experiments. A copy always had to stay in the lab out of fear that the lab books may be needed in a law suit one day.

Grumpiness

I recently discovered that I sometimes behave similarly to Gobind as a graduate advisor. My PhD was not my first research experience. I had spent an entire summer at the Max Planck Institute for plant breeding research in Cologne doing nothing else but repeating the same transformation of a plant plasmid in E. coli cells over and over again. I literally went to the 37 °C incubator room on a daily basis to find that my transformation again didn’t work. My first experiment in Gobind’s lab was a transformation of the pMT4 plasmid carrying the opsin gene. It worked on the first try. I was so happy when I was walking with my plate displaying colonies through the corridor and bumped into Gobind. He asked me grumpily why I was so happy and I told him my transformation had worked. He just looked at me in amazement of how that could possibly make me happy. My current graduate student came to my office jumping up and down of joy because she had figured out what experiment she will do first. I thought it was trivial and looked at her in grumpy amazement.

Asserting independence from others or the artifact finder

A Nobel prize winner’s lab is also not immune to artifact finding. I had the following experience from my Diploma research project at Imperial College. I was worried I would be known as the artifact finder. My project was on cytochrome b559 (the same protein that made me interested in conformational changes that made me become interested in Gobind’s lab). Its proposed role was in protection against damage caused by high light intensities (Barber and De Las Rivas 1993), called photoinhibition, a hypothesis published in PNAS, a journal with impact factor 9.7. During the course of 1 year, I proved beyond a reasonable doubt that the crucial experiment that lead to this exciting hypothesis, was indeed an artifact brought about by the addition of an enzyme cocktail used to keep the reaction conditions anaerobic without the need for otherwise cumbersome inert atmosphere maintenance equipment. However, one of the enzymes added had a chromophore that was in fact absorbing light, and in its light-activated state transferring an electron to cytochrome b559, resulting in its reduction. This reduction had been assumed to be mediated by cytochrome activation itself and the finding of this additional enzyme involvement was not a welcome, but rather boring, finding. The journal that my findings were published in is now discontinued (Klein et al. 1995) and a specialized journal with impact factor 3.1 (De Las Rivas et al 1995).

When I first joined Gobind’s lab, it wasn’t at all clear what exactly I would be working on (despite my enthusiasm about conformational changes). I was first made to repeat some standard experiments to purify rhodopsin to learn the basic techniques. In addition, I discussed projects with one of the postdocs in the lab. He showed me the graph shown in Fig. 9 for a rhodopsin mutant associated with the retinal degeneration disease, retinitis pigmentosa. It looked fascinating — the rhodopsin mutant seemed to be an oscillating protein? After shining light on the cuvette, the intensity at 500 nm dropped to zero and the intensity at 380 nm increased by a comparable amount as expected. However, subsequently a part of the 500 nm recovered, and 380 nm dropped. I jumped on this study with excitement and decided to first repeat the experiment with the wild-type protein I had just prepared. It showed the same behavior. That made me suspicious — rhodopsin had been studied for decades and no one had observed these oscillations. More careful inspection revealed that the size of the fiber optic tip used to bleach the rhodopsin was small compared to the size of the cuvette and that portions of the samples had not been bleached; through convection unbleached and bleached portions of rhodopsin became mixed, giving the oscillatory behavior (Fig. 9). At that moment, I remembered my oath to not become an artifact finder. I decided that I wanted to work on real biological questions, or at least find my own artifacts in the future. I carved out my own project on NMR with rhodopsin. NMR with a membrane protein was challenging, but I was still able to make interesting discoveries that were semi-quantitative descriptions of conformational changes, rather than actual three-dimensional structures, but at least a step in the direction I set out when I first laid eyes on Gobind’s paper on conformational changes in bacteriorhodopsin in the library in Cologne.

Fig. 9.

Bleaching and seemingly recovery of chromophore by absorbance spectroscopy. (Photograph used with permission of the owner, Judith Klein-Seetharaman)

Gobind’s influence on my life carries on

The reviews of my very first NIH RO1 proposal stated “if only this PI wasn’t working on rhodopsin, this would be an interesting grant.” Many times have I tried to leave rhodopsin behind and work on other proteins, thousands of other proteins in fact through computational systems biology, but I have always returned to rhodopsin. We recently worked on corals, and in my desperation to understand anything about this topic, I looked for rhodopsins in the coral genome. I found four of them (Fig. 10). We are now in the process of studying them in the lab. It appears to be my fate: most recently I joined Prof. Petra Fromme’s Center for Structural Discovery. Every lab room, every instrument room, including the one housing a synchrotron has three light switches, controllable with a key, to switch between white light, green light and red light. This is because she is famous for her work on photosynthesis. She also collaborates with Michael Brown on rhodopsin, who had offered me a postdoc while I was finishing my PhD with Gobind. At that time, I wasn’t ready to move to the desert yet. Who would have thought I’d end up here? My scientific journey, like the visit to the Grand Canyon while a PhD student with Gobind, has reached full circle: after many deviations I end up close the Grand Canyon and working yet again on rhodopsin. Even though I have worked on many different questions in many different areas of research, I have always come back to the topic of my thesis — rhodopsin. The education I received with Gobind still remains the deepest running expertise I have — I still know many of the identities of the amino acids and their positions crucial to rhodopsin function, and it is the one topic I have never felt shallow about. Thus, the work with rhodopsin continues, now with coral opsins (Fig. 10). Thank you Gobind. I wish we could write paper number X in the series of structure and function in rhodopsin together.

Fig. 10.

Gobind’s legacy: rhodopsin research in my lab continues. A Homology models of coral opsin candidates (Kumar et al. 2023). B Experimental setup in my lab with spectroscopy setup (left), biochemical preparation (center) and coral tank (right). (Photograph used with permission of the owner, Judith Klein-Seetharaman)

Author contribution

The article was written entirely by the author.

Data availability

Not applicable.

Code availability

Not applicable.

Declarations

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

Not applicable.