Split-protein systems: beyond binary protein–protein interactions

Abstract

It has been estimated that 650,000 protein-protein interactions exist in the human interactome [1], a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, E. coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches.

Rational design of a split-Cas9 enzyme complex

“…Split-protein systems have often been designed such that the functional unit is restored through the interactions of an exogenous pair of proteins (14). For example, DNA-binding and effector domains of modified TALEs have been fused to CRY2 and CIB1 to enable light-inducible control of gene expression (15).…”

Supramolecular Control over Split‐Luciferase Complementation

“…Q8 has demonstrated significant potential as a scaffold for the formation of supramolecular protein complexes,19, 20 as well as to modulate the function of biomaterials 21, 22, 23. Here Q8 is used to reconstitute a split‐protein system, split‐luciferase,24 via selective stabilization of the native split‐protein heterocomplex, enabling reversible signal generation, a large dynamic range, and a generic approach to control protein activity in an in vitro setting (Scheme 1). …”

Nanoparticle mediated delivery and small molecule triggered activation of proteins in the nucleus

“…To be able to quantify the rate and efficiency of delivery we used a split fluorescent protein (Venus) which is only one of a rapidly growing list of proteins that can be split and then functionally complemented. Therefore, this approach could easily be extended to a large number of proteins through either trial and error or based on structural information and should be applicable to single globular domains as well as multidomain proteins [45,46]. …”