Primary goals of the post-genomic era are the assignment of functions to each of the genes encoded by a given genome, and their integration into metabolic and regulatory networks. While transcriptomics and proteomics are progressing rapidly, the collection of other essential information for building these network maps — the mapping of protein activities and of the small molecule intermediates and ions they act on — is being developed. Analyses that are based on mass spectrometry provide a glimpse of the total metabolite and ionic inventory, but lack the temporal and spatial resolution that are necessary to provide local concentrations and flux rates in vivo
. Classical biochemical approaches are very useful in quantifying metabolite levels and in determining when a protein is made and where a protein might reside in the cell at any given time. However, these approaches are extremely limited in their ability to resolve where and when a protein is active, or when and where it interacts with other proteins or with their substrates. Such detailed spatial and temporal information is essential for building realistic network maps, particularly as proteins, metabolites and ions might simultaneously fulfill diverse roles in multiple pathways, requiring the tight control of the timing and location of their activity [1••
]. Understanding homeostasis, metabolic regulation, and cellular signaling thus requires novel concepts and technologies.
Fluorescent ligands and proteins have revolutionized bioimaging, and comprise a new set of tools for addressing cell biological questions at the systems biology level. The ability to genetically encode fluorophores, such as the Aequorea victoria
green fluorescent protein (GFP), has yielded significant advantages for in vivo
studies. These include the capacity to introduce probes into a wide variety of organisms and/or cell types, to control the timing and level of probe expression, and to target probes to specific cellular compartments. Perhaps most importantly, the genetic encoding of optical probes enables methods for high-throughput analysis of protein and cell function [2••
]. The development of a large number of novel fluorescent proteins (FPs), which might be derived from corals and anemones or generated by mutagenesis, has accelerated the development of the fluorophore tool kit by providing the researcher with a selection of spectral variants and fluorophores that have useful properties such as photoconversion [3
Genetically encoded fluorophores offer several types of tools that can be used to probe molecular behavior in living cells. As simple fusion tags, FPs can help to identify protein localization and dynamics. Split FPs can be used to test for protein–protein interactions in vivo
() and to create small peptide tags. Peptides that translocate in response to cellular signals and modification can be used to assay metabolites and protein activity [10••
]. Finally, the (in vivo
) combination of FPs with a second fluorophore provides novel applications that rely on fluorescence resonance energy transfer (FRET). FRET detects rearrangements of the relative orientation and distance of two fluorophores within the 1–10 nm range, thus providing extraordinary spatial resolution and vast possibilities for measuring protein–protein interactions or the creation of small molecule biosensors. Here, we review recent advances in the development and application of genetically encoded biosensors, and discuss how they might be applied to extend our understanding of metabolic and regulatory networks through novel high-throughput analyses.
Figure 1 Fluorescent biosensors for protein–protein interactions. Models for protein–protein interaction biosensors using (a) full-length fluorescent proteins or (b) the split-GFP to reconstitute fluorescent proteins with different spectral properties. (more ...)