A computational method is proposed for inferring protein interactions from genome sequences on the basis of the observation that some pairs of interacting proteins have homologs in another organism fused into a single protein chain. Searching sequences from many genomes revealed 6809 such putative proteinprotein interactions in Escherichia coli and 45,502 in yeast. Many members of these pairs were confirmed as functionally related; computational filtering further enriches for interactions. Some proteins have links to several other proteins; these coupled links appear to represent functional interactions such as complexes or pathways. Experimentally confirmed interacting pairs are documented in a Database of Interacting Proteins.The lives of biological cells are controlled by interacting proteins in metabolic and signaling pathways and in complexes such as the molecular machines that synthesize and use adenosine triphosphate (ATP), replicate and translate genes, or build up the cytoskeletal infrastructure (1). Our knowledge of proteinprotein interactions has been accumulated from biochemical and genetic experiments, including the widely used yeast two-hybrid test (2). Here we ask if protein-protein interactions can be recognized from genome sequences by purely computational means.Some interacting proteins such as the Gyr A and Gyr B subunits of Escherichia coli DNA gyrase are fused into a single chain in another organism, in this case the topoisomerase II of yeast (3). Thus, the sequence similarities of Gyr A (804 amino acid residues) and Gyr B (875 residues) to different segments of the topoisomerase II (1429 residues) might be used to predict that Gyr A and Gyr B interact in E. coli.To find other such putative protein interactions in E. coli, we searched the 4290 protein sequences of the E. coli genome (4) for these patterns of sequence homology (5). We found 6809 pairs of nonhomologous sequences, both members of the pair having significant similarity (6) to a single protein in some other genome that we term a Rosetta Stone sequence because it deciphers the interaction between the protein pairs. The 4290 proteins could form at most (4290) 2 /2 ϭ 9 ϫ 10 6 pair interactions, but we would expect many fewer interactions in a functioning cell; roughly 2 to 10 interactions for each protein does not seem unreasonably many. Each of these 6809 pairs is a candidate for a pair of interacting proteins in E. coli. Five such candidates are shown in Fig. 1. The first three pairs of E. coli proteins were among those easily determined from the biochemical literature in fact to interact. The final two pairs of proteins are not known to interact. They are representatives of many such pairs whose putative interactions at this time must be taken as testable hypotheses.We devised three independent tests of interactions predicted by the method we term domain fusion analysis, each showing that a reasonable fraction may in fact interact. The first method uses the annotation of proteins given in the SWISS-PROT database (7). For cases wh...

Summary Fluorescent proteins have become valuable tools for biomedical research as protein tags, reporters of gene expression, biosensor components, and cell lineage tracers. However, applications of fluorescent proteins for deep tissue imaging in whole mammals have been constrained by the opacity of tissues to excitation light below 600 nm, due to absorbance by hemoglobin. Fluorescent proteins that excite efficiently in the “optical window” above 600 nm are therefore highly desirable. We report here the evolution of far-red fluorescent proteins with peak excitation at 600 nm or above. The brightest one of these, Neptune, performs well in imaging deep tissues in living mice. The crystal structure of Neptune reveals a novel mechanism for red-shifting involving the acquisition of a new hydrogen bond with the acylimine region of the chromophore.

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.