Constructing and exploiting the fluorescent protein paintbox (Nobel Lecture).

My first exposure to visibly fluorescent proteins (FPs) was near the end of my time as a faculty member at the University of California, Berkeley. Professor Alexander Glazer, a friend and colleague there, was the world s expert on phycobiliproteins, the brilliantly colored and intensely fluorescent proteins that serve as light-harvesting antennae for the photosynthetic apparatus of blue-green algae or cyanobacteria. One day, probably around 1987–1988, Glazer told me that his lab had cloned the gene for one of the phycobiliproteins. Furthermore, he said, the apoprotein produced from this gene became fluorescent when mixed with its chromophore, a small-molecule cofactor that could be extracted from dried cyanobacteria under conditions that cleaved its bond to the phycobiliprotein. I remember becoming very excited about the prospect that an arbitrary protein could be fluorescently tagged in situ by genetically fusing it to the phycobiliprotein, then administering the chromophore, which I hoped would be able to cross membranes and get inside cells. Unfortunately, Glazer s lab then found out that the spontaneous reaction between the apoprotein and the chromophore produced the “wrong” product, whose fluorescence was red-shifted and fivefold lower than that of the native phycobiliprotein. An enzyme from the cyanobacteria was required to insert the chromophore correctly into the apoprotein. This enzyme was a heterodimer of two gene products, so at least three cyanobacterial genes would have to be introduced into any other organism—not counting any gene products needed to synthesize the chromophore. Meanwhile, fluorescence imaging of the second messenger cAMP (cyclic adenosine 3’,5’-monophosphate) had become one of my main research goals by 1988. I reasoned that the best way to create a fluorescent sensor to detect cAMP with the necessary affinity and selectivity inside cells would be to hijack a natural cAMP-binding protein. After 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 subunits: regulatory and catalytic. In the absence of cAMP, the regulatory subunits tightly bind and inhibit the catalytic subunits. When cAMP becomes available, it binds to the regulatory subunits, which then let go of the catalytic subunits, which in turn start transferring phosphate groups from ATP onto selected proteins. But how could activation of PKA by cAMP be made directly visible inside a single living cell? From my graduate student days I had been fascinated by a biophysical phenomenon called fluorescence resonance energy transfer (FRET), in which one excited dye molecule can transfer its energy to a close neighbor, much as a football or basketball player can pass the ball to a team mate—with diminishing probability of success the greater the distance between the players. If we could attach one type of dye molecule to the regulatory subunits and the other type of dye molecule to the catalytic subunits, FRETwould be possible in intact PKA, because the subunits are in intimate contact. But once cAMP had broken up the PKA complex and allowed the subunits to drift apart, FRET would be disrupted and a change in fluorescence color should be observable. But to get these experiments to work, we needed abundant supplies of PKA subunits and lots of advice on how to handle them, especially because we had very little experience with protein biochemistry. I contacted Susan Taylor, who had become one of the world s leading experts on PKA and was producing relatively large quantities of recombinant PKA subunits in order to solve their crystal structure (Figure 1). The Taylor lab kindly sent shipment after shipment of proteins on wet or dry ice from UCSD to Berkeley for us to try to label with dyes, but the dyes either refused to stick or messed up the subunits to the point where they would no longer respond to cAMP. The wish to facilitate this collaboration was an important part of the reason that my lab moved from Berkeley to UCSD in 1989. Eventually, after a year of working side by side, Dr. Stephen Adams in my lab and Ying Ji Buechler and Wolfgang Dostmann in the Taylor lab devised a reproducible procedure to combine fluoresceinlabeled catalytic subunits with rhodamine-labeled regulatory subunits to produce FRET-based sensors for cAMP. Over the next few years, we used these protein complexes to study several interesting problems in cAMP signaling. For example, we collaborated with Eric Kandel s lab to demon-