Last year’s chemistry Nobel Prize was one of the most softball predictions ever made for the Nobel Prize. The Green Fluorescent Protein (GFP) has become so widely used in chemistry, biology and medicine that it is easy to forget that someone had to discover it and develop the technology. Every year Roger Tsien’s name used to be on everybody’s favorite candidate list along with Martin Chalfie’s and Osamu Shimomura’s. Then last year, he along with Shimomura and Chalfie finally put the tortuous process and spilling of ink to rest.
A post about GFP is a writer’s dream for indulging in pretty pictures. I will restrict myself to two. GFP has become a poster boy for the science of biotechnology. Its barrel shaped ß-sheet structure shown above has become iconic in the scientific world. This is most emblematic in the odd and many varieties of glowing animals that now grace the covers of everything from scientific journals to websites and children’s textbooks. If as some have predicted, we happen to “domesticate” biotechnology in the next few decades, it is very likely that one of the first things that our children would do would be to produce glowing pet rabbits, dogs, mice and cats. Along with a few other icons like DNA and the fruit fly, the image of glowing animals and fluorescent proteins is now deeply ensconced in our imagination as an example of what humans can do by manipulating biological systems. Perhaps one day our children can become friends with transgenic, green, glowing human beings, without the hulk-like physique and temper tantrums.
Osamu Shimomura’s talk is essentially historical. He starts by reviewing different types of fluorescent reactions in nature. These include the luciferin-luciferase system used by fireflies. Luciferin is a small molecule that gets decarboxylated by luciferase in the presence of oxygen resulting in bioluminescence. Depending on the products formed you can get red or yellow-green luminescence. Bioluminiscence is also observed in coelenteran organisms including species of jellyfish and shrimp. The coelenteran luminescence involves a different kind of reaction in which ring opening of a heterocyclic compound leads to an acyl amino pyrazine derivative. The light emitter consists of the form of the acyl amino pyrazine in which the amide nitrogen is deprotonated. Another example of bioluminescence takes places in bacteria. In this case luciferin itself is oxidized to a light-emitting compound. The reaction takes place in the presence of the cofactor FMN. Shimomura illustrated further examples of luminescence involving shrimps, earthworms, and dinoflagellates. The operative elements in every case are molecular oxygen and luciferase.
A particularly striking example of bioluminescence concerns Aequorin, a small photoprotein in the jellyfish Aequoria victoria. In the presence of calcium ions another ring opening reaction takes place that produces blue fluorescence. Because of its dependence on calcium the reaction is a valuable one for measuring intracellular calcium levels.
Luminous fungi and mushrooms are well-known in nature, frequently emitting green light. The reaction in case of luminous fungi involves a precursor that in the presence of methylamine possibly oligomerizes to a trimer. Reaction with superoxide, oxygen and a cationic detergent-like small molecule tetradecanoylcholine produces the light emitting compounds. The reaction is widely observed in several species of mushroom.
Interestingly, Shimomura stated that most of the above findings were made before the 1980s. GFP and the revolution came later, but it would not have been possible if the foundation for understanding and studying bioluminescence had not been previous laid.
Martin Chalfie starts by noting that the winning the Nobel Prize got him Andy Warhol’s 15 minutes of fame. He showed us a Google news snapshot taken the day he won the prize which demonstrated that he had finally made it into the rarefied heights of the land of celebrities, very much in the same league as Britney Spears and Amy Winehouse.
Chalfie’s major field of research now is not GFP but research into how animals sense various signals in their environment. Proprioception or the sense of touch is a particular interest, exemplified by Chalfie’s research on C. elegans, perhaps the only worm in history that has not been universally despised.
However some retelling of history was in order. Chalfie recounted how Douglas Prasher sent them the clone for GFP in the late 1980s. GFP effects a five-membered ring formation reaction when irradiated with UV light. Only a few months after getting the clone, a new graduate student in Chalfie’s used the fluorescent microscope in her old lab (realizing that the microscope in Chalfie’s lab was a piece of junk) and made fluorescent bacteria for the first time. The study made it to the cover of Science and was supposed to say “Green Fluorescent Protein” A New Marker of Gene Expression”. The Science editors disapproved of the title, saying that every paper published in science has something “new” to say. Another title was rejected as being “too technical”. The title was finally modified and accepted after getting rid of the word “new”.
Incidentally Chalfie used some unpublished results that his wife, who was also a biochemist, was working on. In return Chalfie had to promise his wife in writing that he would make coffee, cook her a French dinner and take out the garbage every night. This is one important take-home message for students and future scientists; science can involve some radical compromises with life.
GFP became enormously influential. Roger Tsien improved the properties of GFP by modifying its spectral characteristics. The protein has now become a ubiquitous tool in studying gene expression in an astonishing variety of scientific studies. Now, almost twenty years after Chalfie’s pioneering work, there are about 30,000 papers in the literature citing the use of GFP.
Chalfie’s current research concerns how organisms sense mechanical signals in the environment. His group is trying to identify the genes that are key in producing the sense of touch. Apart from other techniques, his group is using RNA interference to silence possible genes for touch and investigate the effect of doing this. Feeding RNAi to C. elegans downregulates genes in muscles, skin, intestine and the germline. It is difficult to investigate the exact effect of doing this, however, because it results in the animals’ death. A protein called SID-1 in neurons as well as integrins seem to be involved in the sense of touch. Chalfie’s group has discovered an unusually large number of genes involved in proprioception. The functions of most of these genes are unknown and therefore the future looks exciting and challenging.
Chalfie ends with a couple of important lessons about science:
1. Science is cumulative.
2. The students and postdocs are the real innovators in science. Many times it is easy to forget that they are the originator of some of the key details of Nobel Prize-winning ideas.
3. Basic research is essential and is the engine that drives innovation. Science cannot be done with too specific goals in mind.
4. All life should be studied, not just model organisms.
Each one of these points is worth contemplating. Freeman Dyson once said that scientists can be divided into two groups; birds who soar above the skies and survey the grand scientific landscape, and frogs who play around in the mud and discover interesting details. Frogs may not always win Nobel Prizes, but without their work the birds would not have a scientific landscape to survey. The graduate students and postdocs who contribute to Nobel Prize winning research may be the ultimate frogs, and their importance cannot be underestimated. The point about cumulative results is also very important. Breakthroughs in science are built on years of quiet, behind the scenes, careful and patient work. Here again, the work done by the playful frogs builds up to the insights gained by the high-flying birds, and this work is really the engine that drives science. And the quip about studying all life and not just model organisms brings a quote by the great organic chemist R. B. Woodward to mind; Woodward said that the best model for a molecule is the molecule itself. Similarly, only life can serve as the idea model for studying life.
Roger Tsien channeled his childhood love of bright colors into a Nobel Prize. He begins by giving a perspective on creativity in analysis versus synthesis. Synthesis has been the traditional and unique domain of chemistry. Some of the most important advances in medicine in the last year have come from the application of synthesis to interesting biological problems. Synthesis is akin to architecture or sculpture on a molecular scale.
Tsien talks a little about his own background. He is the youngest of 3 brothers and comes from a family of engineers and wanted to find a unique ‘ecological’ niche for himself. Charles Darwin was another youngest child, and he did manage to carve his own niche. Tsien was fascinated by colors from an early stage and became interested in biology and chemistry. One of the key points emphasized by Tsien was that young researchers should explore interdisciplinary areas where traditional barriers may prevent the transfer of knowledge.
Tsien’s approach is to look at a “big” biological problem and ask if and how it can be approached at a molecular level. Tsien’s original interest was in monitoring and interpreting intracellular levels of calcium. Calcium is universal and key in many processes of life. Measurement of calcium levels was a significant challenge since calcium-mediated signaling is extremely fast and calcium levels are very low. In addition one has to detect low concentrations of calcium in the presence of a very large background of magnesium. Tsien began by investigating chelators which would demonstrate selectivity for calcium over magnesium. The common chelator EDTA had 5-fold selectivity for calcium and was a good starting point, but it did not have spectral properties. Tsien started putting phenyl rings in various places in the molecule for endowing it with color properties. The molecule also had to be stable at different pH values, and simple chemical considerations dictated modifications that would do this. Tsien finally obtained a molecule that was UV sensitive and bound calcium selectively.
However the resulting molecule had four negative charges and was not very cell permeable. The negative charges were necessary for chelating calcium, but Tsien realized that the negative groups needed to be unmasked only inside the cell. To get to a cell permeable compound Tsien used one of the oldest and most common strategies in the organic chemist’s bag of tricks; protecting the negative acid groups by attaching appropriate hydrophobic ester groups to them that would increase permeability through the hydrophobic lipid membrane. The ester groups would disintegrate inside the cell, exposing the essential molecular structure for binding calcium.
One of the first and most important applications of such molecules was observing the calcium wave during fertilization of en egg with sperm. Tsien then turned his attention to the prototypical second messenger, namely cyclic AMP. cAMP is a ubiquitous intracellular messenger and tracking its movement would be invaluable for all kinds of studies. Tsien wanted to image cAMP in the complex environment of the cell. He decided to use a natural cAMP sensor, protein kinase A (PKA) whose activity is intimately coupled with that of cAMP. Tsien applied the then new method of fluorescent resonance energy transfer to investigate this process and obtained exciting results.
It was then (1992) that Tsien heard about Douglas Prasher’s cloning of the GFP gene. But there were problems with wild-type GFP. It had a very broad acceptor spectrum precluding its use as a FRET acceptor. Since GFP’s properties as an acceptor depend on the exact arrangement of amino acids in its structure and having knowledge of the kind of reactions GFP undergoes, Tsien decided to mutate certain amino acids in order to shift he absorption spectrum to particular wavelengths. Initial attempts at prediction utterly failed, but a conservative substitution of a threonine group for a serine group turned out to be the key to improving the spectrum. At the same time a crystal structure of GFP was obtained which corroborated the design strategy.
Initial attempts at publication in Science failed, with one referee raising the rather silly objection that the crystal structure of the protein gave no indication of its ecological role. Only the possibility of getting scooped in another journal could convince the Science editors to accept the paper.
Tsien then mentions some of the limitations of fluorescent proteins. They can be too big and their excitation wavelengths may not penetrate tissue. Plus the obvious scientific and moral problems associated with effecting gene transfer of GFP into humans are apparent. Magnetic Resonance Imaging is frequently required to probe the details of GFP effects. Efforts are underway to circumvent these problems.
Tsien concludes by talking a little about his current research which involves investigating metastasis. The work includes imaging tumors and nerves with GFP. Green tumors are easier to visualize and remove with surgery than non-fluorescent tumors. At the same time nerves should not be removed, and labeling them with a different color helps to avoid this. Imaging techniques provide a very sensitive technique for surgeons to distinguish unwanted tumors from necessary nerve growth. Tsien ends with a spectacular image of a plate displaying bacteria that have been colored by different fluorescent protein expression.
Tsien, Chalfie’s and Shimomura’s talks held many notable lessons. You should take risks and work on big problems; even if you don’t succeed at least you would have tried and learnt something. Persistence pays off. Don’t give up if your predictions fail. Your papers may not get accepted by top journals…or sometimes may get accepted for the wrong reasons. Find good collaborators and be kind to them (Tsien confesses that he was personally incompetent in molecular biology techniques). And finally, always remember that luck plays a significant role in discovery, but don’t depend on it. Do science for the pleasure of finding things out, for the kick that you get from the joy of discovery. Revel in it, savor it, because in the end that will be the only thing that really matters.