Systematic control of protein interactions for systems biology.

S ince its birth, systems biology has gained a great deal from the protocols devised to study phenomena at the level of single proteins and nucleic acids. Such protocols find broad markets and utility at higher levels of biological organization, from next-generation sequencing, which uses modified nucloeotides and fluorescent identifiers (1), to ChIP-seq analysis, which identifies histone modifications and binding sites in protein–DNA interactions (2). In PNAS, Sivaramakrishnan and Spudich introduce a system for interrogating interactions between pairs of proteins, domains, and peptides (3), and it is very possible that their invention will find applicability in the construction and analysis of large-scale protein–protein interaction networks (4). The technology devised by Sivaramakrishnan and Spudich is based on Forster resonance energy transfer (FRET), which relies on the proximity-dependant excitation of an acceptor molecule by a donor (5). This excitation may be detected with spectral imaging, so FRET enables the investigator to directly probe interactions between candidate pairs of biomolecules, by using molecular cloning to tether the donor to one candidate and the acceptor to the second. For years this technology has enjoyed broad applicability (6–8), and biosensors using FRET have been applied in live cell imaging to investigate an array of biological processes, such as phosphorylation, protease activity, fluctuations in membrane potential, and changes in redox potentials, pH, and other environmental conditions (9, 10). To engineer the effective concentrations of interacting species, Sivaramakrishnan and Spudich have recruited the ER/K single α-helix as an intradomain and intraprotein linker, in conjunction with FRET sensing, to quantitatively regulate and study interactions between proteins fused to the ends of the link. The effective concentrations are determined by the size of the linkage. Perhaps not surprisingly, the on-rate of the interaction decreases as the length of the linker increases. There is little effect on the dissociation rate. Importantly, the value of this system lies not only in the ability to modulate the interaction frequencies of two molecules without major perturbations to their structures, but also to obtain meaningful and accurate estimates of the natural binding affinities associated with interactions. Thus, this application has farreaching implications for the large and diverse array proteins for which interactions with other cellular species are intrinsic to their basic functionality. There are several advantages to this linker system. The linkage motif itself has been adopted from naturally occurring proteins, thereby precluding the need of designing the construct ab initio. Second, these biosensor constructs are designed with (Gly-Ser-Gly)4 bridges separating the interacting domains and FRET species, thereby allowing the elements of the construct to fully explore rotational degrees of freedom. The unique linker system has built-in modularity as well; protease sites separate its various domains, thereby lending the system separation and purification of its constituents, which are thus made available in stoichiometric amounts for bimolecular interactions and affinity calculations. The interactions between proteins are inevitably tied to the motions and dynamics of the interaction participants, and a protein’s conformation is an important factor in determining its interaction affinity. The linker system provides several avenues that may be of interest to the study of conformational changes. When Fig. 1. Applying the ER/K α-helix to studying conformational changes and network characterization. (A) A protein in two possible conformations. Detection of a FRET signal may be dependent on a change in conformation. The semicircle below each conformation represents the linker. (B) Two representations of a protein–protein interaction network. Left: This network is a conventional node-and-edge depiction. Quantifying the interaction affinities may enable the addition of interaction strength as a property of the network, yielding a network with weighted edges (Right). Colored interaction schematics above and below the central arrow correspond to examples of mapped highand low-affinity interactions between similarly colored nodes in the affinity-based network.