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Interactive Learning: Lessons from Two Hybrids Over Two Decades

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Interactive Learning: Lessons from Two Hybrids Over Two Decades Proteomics 2009, 9, 5209–5213 DOI 10.1002/pmic.200900236 5209 REVIEW Interactive learning: Lessons from two hybrids over two decades Stanley Fields Howard Hughes Medical Institute, Departments of Genome Sciences and Medicine, University of Washington, Seattle, WA, USA The initial yeast two-hybrid experiment – published in 1989 – described an approach to Received: April 13, 2009 detecting protein–protein interactions that has flourished over the last two decades, leading to Revised: June 10, 2009 the assembly of large-scale data sets of these interactions. Yet the yeast assay originated Accepted: June 13, 2009 because of the laboratory’s interests in technology development, not because of its need to identify partners of any protein then under study. In addition to such motivating forces, other features of the process of originating a technology can be revealed by considering the lessons of the two-hybrid approach. These include the value of timeliness in a method’s development, the willingness of an investigator to try experimental approaches that prove fruitless, the ability of biological macromolecules to display surprising attributes, the benefits of a community expending efforts to expand the uses of a technology platform, and the role of scientific training of those who work in technology. Keywords: Protein arrays / Protein interaction / Technology development / Two-hybrid / Yeast 1 Introduction development that I have carried with me throughout my research career. While 2009 marks two decades since the publication of the Nature paper [1] describing the yeast two-hybrid assay, the point in time when the method came to mind was – perhaps 2 The origins of technology not too surprisingly – 2 years earlier. In the intervening 22 years, the idea has grown from a hand-drawn sketch (Fig. 1) It is often said that new technologies arise in response to a to a staple of biology laboratories, fastening together specific biological problem, but the two-hybrid approach proteins derived from a veritable zoo of organisms. Its refutes the universality of this claim, reflecting a different central principle – that the two functional domains of a reality of how methods often come to be. The yeast assay transcriptional activator can be split apart and each fused to was not developed because of our need to find partners for one of a pair of partner proteins in order to reconstitute the any protein we were studying two decades ago, nor did it activator’s ability to turn on a reporter gene – has spawned a spring from our awareness that a simple method to do so brood of related technologies, as well as displays of cellular did not then exist and had to be devised. In fact, my networks with thousands of interactions, and the algorithms laboratory at the time could not have reasonably been said to to decipher the implications of these networks. The two- even have a focus on protein interactions, although it did hybrid history has also been personally instructive, provid- have an interest in technology. ing lessons about the nature and nurture of technology The immediate motivation for the two-hybrid system was a request for grant applications that would lead to results with commercial potential, a request that would seemingly Correspondence: Dr. Stanley Fields, Howard Hughes Medical Institute, Departments of Genome Sciences and Medicine, be met by a proposed assay system whose use in the biotech University of Washington, Box 355065, Seattle, WA 98195, USA and pharmaceutical industries could be licensed and whose E-mail: fi[email protected] products – plasmids, strains, libraries and other reagents – Fax: 11-206-543-0754 could be sold by companies that supply biomedical research & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 5210 S. Fields Proteomics 2009, 9, 5209–5213 specific knowledge of the structure and modularity of eukaryotic transcription factors. On the other hand, the method fortunately predated the remarkable developments in genome sequencing and in MS instrumentation that enabled the rapid identification of proteins present in a complex by a determination of the sequence of short peptides from these proteins. Had these developments Figure 1. The first sketch of the two-hybrid assay, as provided in already been in play, the yeast method might have had far a grant application submitted at the end of 1987 to the Procter fewer takers. and Gamble Company. The native yeast Gal4 protein is shown Timeliness looms large in most technologies. In the early as having DNA-binding (GAL4D) and activation (GAL4A) days of the yeast method, biochemists often said, perhaps domains, with the DNA-binding domain recognizing a site on ruefully, that yeast two-hybrid took a task – the purification DNA known as the Upstream Activation Sequence for the GAL of protein complexes – that had been the provenance of genes (UASG). Proteins P and Q form hybrids with the DNA- binding and activation domains, respectively, and reconstitute experts and replaced it with another task – a microbial transcriptional activity, leading to expression of the GAL1 gene. genetic selection – that could be carried out by under- graduates new to biology. In the parallel universe of biochemistry, the advent of the TAP tag [2] 10 years ago laboratories. Despite these now obvious means to royalty provided an early means to conveniently purify a protein income, the application outlining what became the two- and its interacting partners. It was this tag, and the many hybrid assay was not funded, a consequence of the review other simple protein purification strategies it spawned, that panel not being persuaded of the technology’s feasibility. ultimately brought the task of purifying protein complexes Yet, in spite of the negative funding news, we immediately into the undergraduate laboratory. Such is always the nature initiated experiments to develop the method, based solely on of important technological discoveries: the unthinkable the beauty and elegance of this idea: if this assay becomes, first, thinkable and then, not too much later, worked, surely it would have tremendous utility to routine. address problems being tackled by biologists. A few months A lesson associated with timeliness is the inter- later, we resubmitted the grant application to Procter and connectedness of technological advances. The yeast two- Gamble’s University Exploratory Research Program, which hybrid assay arrived just as sequencing of whole genomes – funded the application and provided us with a small 3-year of bacteria, then yeast, the nematode, the fruit fly, and the grant. more recent numerous representatives of all the kingdoms In fact, an argument can plausibly be made that it is the of life – began to take off. The ramifications of the questions biologists pose that follow directly from the sequencing revolution for two-hybrid methodology were technologies available to them, rather than the converse. For many: genome sequences enabled the small inserts in two- instance, it makes little sense to purify vanishingly small hybrid libraries to be assigned to proteins; they allowed array quantities of a protein complex if mass spectrometric approaches in which full-length open reading frames fused methods do not exist to identify the constituents; it is not to DNA-binding or activation domains could be made and worth postulating how the expression of thousands of genes analyzed [3, 4]; they galvanized scientists and funding changes in response to a cellular stress if DNA arrays had agencies to consider functional studies on complete sets of not come into fashion; and it is not logical to propose proteins; and they brought in computational biologists to surveys of the entire human genome for variations linked to devise new algorithms, including those that analyzed disease if an extensive set of polymorphisms and the protein interaction data. instrumentation to rapidly detect DNA sequence poly- morphisms are not available. Biologists who do not adjust their thinking – and even more, the aims of their research – 4 The likelihood of failure in response to continuing technological innovations soon find themselves left in the dust of their more intrepid Another enduring lesson came as the realization set in of all colleagues. the reasons why the yeast two-hybrid method might not or even – according to early skeptics – should not work. To name just a few of these reasons: most proteins need not 3 The value of timing have a modular structure that allows the construction of usable activation domain hybrid libraries; the affinity of The two-hybrid method, which followed directly from the most interactions might not be sufficient to trigger tran- state of knowledge about transcription in the mid-1980s, scription in the two-hybrid configuration; most non-nuclear demonstrates the critical nature of timeliness for a new proteins might not be readily directed to the nucleus; and technology. On the one hand, the two-hybrid assay could not the eukaryotic transcription complex might have stereo- have been developed much earlier because it required chemical constraints that require defined protein constructs & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics 2009, 9, 5209–5213 5211 to position the DNA-binding and activation domains in PCR and now high-throughput DNA sequencing. For the precise locations. What we came to appreciate was that two-hybrid method, these adaptations included new selec- undue focus on any of these failure scenarios, most of which tion schemes, improved vectors and strains, ‘‘reverse’’ we considered only well after the assay had become estab- methods that select against interactions, approaches to assay lished, might easily have persuaded us not to embark on the DNA-protein, RNA–protein or small molecule–protein approach. Naivete´ can sometimes be helpful. interactions, strategies that detect protein interactions in More generally, any experiment – if thought about long cellular compartments other than the nucleus, numerous enough – can be construed as highly unlikely to impossible other reporter proteins that can be split into two such as to succeed.
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