Constructing and Exploiting the Fluorescent Protein Paintbox (Nobel Lecture)** Roger Y
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Nobel Lectures R. Y. Tsien DOI: 10.1002/anie.200901916 Green Fluorescent Protein Constructing and Exploiting the Fluorescent Protein Paintbox (Nobel Lecture)** Roger Y. Tsien* fluorescent proteins · in vivo imaging · mutagenesis · Nobel Lecture 1. Motivation by cAMP be made directly visible inside a single living cell? From my graduate student days I had been fascinated by a My first exposure to visibly fluorescent proteins (FPs) was biophysical phenomenon called fluorescence resonance near the end of my time as a faculty member at the University energy transfer (FRET), in which one excited dye molecule of California, Berkeley. Professor Alexander Glazer, a friend can transfer its energy to a close neighbor, much as a football and colleague there, was the worlds expert on phycobilipro- or basketball player can pass the ball to a team mate—with teins, the brilliantly colored and intensely fluorescent proteins diminishing probability of success the greater the distance that serve as light-harvesting antennae for the photosynthetic between the players. If we could attach one type of dye apparatus of blue-green algae or cyanobacteria. One day, molecule to the regulatory subunits and the other type of dye probably around 1987–1988, Glazer told me that his lab had molecule to the catalytic subunits, FRET would be possible in cloned the gene for one of the phycobiliproteins. Further- intact PKA, because the subunits are in intimate contact. But more, he said, the apoprotein produced from this gene once cAMP had broken up the PKA complex and allowed the became fluorescent when mixed with its chromophore, a subunits to drift apart, FRET would be disrupted and a small-molecule cofactor that could be extracted from dried change in fluorescence color should be observable. cyanobacteria under conditions that cleaved its bond to the But to get these experiments to work, we needed phycobiliprotein. I remember becoming very excited about abundant supplies of PKA subunits and lots of advice on the prospect that an arbitrary protein could be fluorescently how to handle them, especially because we had very little tagged in situ by genetically fusing it to the phycobiliprotein, experience with protein biochemistry. I contacted Susan then administering the chromophore, which I hoped would be Taylor, who had become one of the worlds leading experts on able to cross membranes and get inside cells. Unfortunately, PKA and was producing relatively large quantities of Glazers lab then found out that the spontaneous reaction recombinant PKA subunits in order to solve their crystal between the apoprotein and the chromophore produced the structure (Figure 1).[8,9] The Taylor lab kindly sent shipment “wrong” product, whose fluorescence was red-shifted and after shipment of proteins on wet or dry ice from UCSD to fivefold lower than that of the native phycobiliprotein.[1–3] An Berkeley for us to try to label with dyes, but the dyes either enzyme from the cyanobacteria was required to insert the refused to stick or messed up the subunits to the point where chromophore correctly into the apoprotein. This enzyme was they would no longer respond to cAMP. The wish to facilitate a heterodimer of two gene products, so at least three this collaboration was an important part of the reason that my cyanobacterial genes would have to be introduced into any lab moved from Berkeley to UCSD in 1989. Eventually, after other organism—not counting any gene products needed to a year of working side by side, Dr. Stephen Adams in my lab synthesize the chromophore.[4] and Ying Ji Buechler and Wolfgang Dostmann in the Taylor Meanwhile, fluorescence imaging of the second messen- lab devised a reproducible procedure to combine fluorescein- ger cAMP (cyclic adenosine 3’,5’-monophosphate) had labeled catalytic subunits with rhodamine-labeled regulatory become one of my main research goals by 1988. I reasoned subunits to produce FRET-based sensors for cAMP.[10,11] Over that the best way to create a fluorescent sensor to detect the next few years, we used these protein complexes to study cAMP with the necessary affinity and selectivity inside cells several interesting problems in cAMP signaling.[12,14] For would be to hijack a natural cAMP-binding protein. After example, we collaborated with Eric Kandels lab to demon- much consideration of the various candidates known at the time, I chose cAMP-dependent protein kinase, now more commonly abbreviated PKA. PKA contains two types of [*] Prof. R. Y. Tsien subunits: regulatory and catalytic. In the absence of cAMP, Howard Hughes Medical Institute and Departments of Pharmacology and Chemistry & Biochemistry the regulatory subunits tightly bind and inhibit the catalytic University of California, San Diego subunits. When cAMP becomes available, it binds to the 9500 Gilman Drive, La Jolla, CA 92093-0647 (USA) regulatory subunits, which then let go of the catalytic subunits, E-mail: [email protected] which in turn start transferring phosphate groups from ATP [**] Copyright The Nobel Foundation 2008. We thank the Nobel onto selected proteins.[5–7] But how could activation of PKA Foundation, Stockholm, for permission to print this lecture. 5612 www.angewandte.org 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2009, 48, 5612 – 5626 Angewandte Fluorescent Proteins Chemie fully assimilated the DNA. Once in the cells, these genes would hopefully make composite proteins in situ that would both fluoresce and preserve native biological function. Knowing that Professor Glazers lab was still struggling with the phycobiliprotein approach, I sought simpler alter- native fluorescent proteins. I remembered that certain jelly- fish (Figure 2) contained a green fluorescent protein (GFP), Figure 1. Schematic representation showing how cAMP-induced disso- ciation of regulatory from catalytic subunits of protein kinase A (PKA) can be reported by loss of FRET from fluorescein to rhodamine labels. Picture: S. Adams, Y. Buechler, S. Taylor. strate spatial gradients of cAMP within individual sea slug (Aplysia californica) neurons undergoing training procedures in a model of synaptic plasticity.[15] Although the cAMP sensor was moderately successful, the general approach would have been very difficult to extend to other proteins because it required high-level expression and purification of soluble proteins or subunits, controlled attachment of two different dyes in vitro to distinct domains or subunits without destroying the function of the protein, Figure 2. The jellyfish Aequorea victoria (or Aequorea aequorea) from repurification, and microinjection back into living cells. Such which aequorin and green fluorescent protein were isolated. Photo cells had to be large and robust enough to tolerate poking courtesy of Dr. Claudia E. Mills, Friday Harbor Laboratories. with a hollow glass needle, and the experimenter had to be patient and dexterous, unlike me. All of the above obstacles because it was an annoying contaminant that needed to be could be circumvented if we had genes encoding two carefully separated away from aequorin, the jellyfish protein fluorescent proteins of the appropriate colors. These genes that some of our competitors used to measure calcium signals could be fused to the genes for the protein(s) of interest. One inside cells.[16] One day in April or May 1992, I typed “green would still have to get the fusion genes into the cell(s) to be fluorescent protein” into Medline, the National Library of studied, but standard methodology has been worked out for Medicines computerized literature searching tool that had most cells of interest. Introducing genes into cells (trans- recently become accessible from UCSD. To my astonishment, fection) is generally much easier than introducing proteins, up popped a citation to a just-published paper by Douglas because each cell needs only one or a few copies of DNA Prasher and collaborators,[17] reporting the cloning of the gene (compared to billions of molecules of protein), the cell has for GFP and the likelihood that the chromophore was an plenty of time to recover from any membrane damage, and integral part of the protein rather than being an external one can selectively propagate those cells that have success- cofactor. I rushed to the library to photocopy the paper (Figure 3), which conveniently listed Prashers telephone Roger Tsien was born in 1952 in New York. number at the Woods Hole Oceanographic Institution. I He studied chemistry and physics at Harvard phoned Prasher in May 1992 and was surprised to hear that he University (BSc 1972) and completed his did not intend to work on GFP any more. En route to the full- PhD in 1977 with R. Adrian at Cambridge length gene, he had first obtained an approximately 70% University. In 1981 he became Assistant Professor at the University of California in length gene and tried to express it into bacteria, but no Berkeley. Since 1989 he has been Professor fluorescence appeared. Because of funding difficulties and a of Pharmacology, Chemistry, and Biochem- change in career direction, he was willing to pass the baton to istry at the University of California in San us, provided that we promised to include him as a co-author if Diego and researcher at the Howard we succeeded. I agreed to this very reasonable request, so he Hughes Medical Institute. His current promised to send us a sample of the DNA-encoding GFP, and research focuses on the imaging and treat- some frozen jellyfish tissue in which we might hunt for the ment of cancer. enzyme(s) that we both feared would be necessary to Angew. Chem. Int. Ed. 2009, 48, 5612 – 5626 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 5613 Nobel Lectures R. Y. Tsien was a huge variation in the brightness of individual cells in the population. Heim and I showed Emr the cells under the microscope and asked if he could suggest any biological question in yeast for which GFP could help supply the answer.