Computation-guided optimization of split protein systems

Abstract

25Splitting bioactive proteins, such as enzymes or fluorescent reporters, into conditionally reconstituting 26 fragments is a powerful strategy for building tools to study and control biochemical systems. However, split 27 proteins often exhibit a high propensity to reconstitute even in the absence of the conditional trigger, which 28 limits their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific, or 29 often ineffective. Here, we report a computational design-driven strategy that is grounded in fundamental 30 protein biophysics and which guides the experimental evaluation of a focused, sparse set of mutants-31 which vary in the degree of interfacial destabilization while preserving features such as stability and catalytic 32 activity-to identify an optimal functional window. We validate our method by solving two distinct split 33 protein design challenges, generating both broad insights and new technology platforms. This method will 34 streamline the generation and use of split protein systems for diverse applications. 35 36 KEYWORDS: synthetic biology, split proteins, computational protein design, protein engineering 37 38

Elucidation and refinement of synthetic receptor mechanisms

“…We next investigated whether the observed effects of TMD choice might extend to synthetic receptors that operate by a distinct mechanism. To test this, we employed a recently reported MESA system in which ligand binding induces reconstitution of a mutant split TEVp (26). The reconstituted protease then releases a TF from the receptor, enabling the TF to enter the nucleus and drive reporter expression ( Fig.…”

“…A third strategy is to employ orthogonal systems with mechanisms that are essentially independent of endogenous signaling and regulation. Examples of this approach include synthetic Notch (synNotch) receptors, which sense surface-bound ligands (21)(22)(23)(24), and the modular extracellular sensor architecture (MESA), which detects soluble ligands (25)(26)(27)(28). Both of these receptor systems can regulate either endogenous or exogenous genes directly through ligand binding-induced release of a transcription factor (TF).…”

“…This study focuses on exploring and improving MESA receptors--a class of self-contained synthetic receptors that signal in a manner that is independent of endogenous cellular pathways (25)(26)(27)(28). Each MESA receptor comprises two transmembrane chains that dimerize upon ligand binding, triggering an intracellular proteolytic trans-cleavage reaction that releases an initially sequestered TF (Fig.…”

“…In this investigation, we build mechanistic understanding and improve the functional performance of MESA receptors by systematically exploring variations upon MESA receptor design that we hypothesized might overcome the aforementioned limitations. We identify several features that may be rationally modified to tune performance (i.e., design handles), and we employ this knowledge to generate new high-performing receptors, expand the MESA repertoire to sense new ligands, and enhance a recently developed MESA variant that employs a distinct mechanism (26). Finally, we synthesize these observations to propose a framework of biophysically motivated design considerations for building novel synthetic receptors.…”

Scalable recombinase-based gene expression cascades

“…We observed a minor (<10%) decrease in %GFP+ cells and premature mCherry expression in the absence of ABA. Point mutations that mitigate the tendency of split recombinase fragments to spontaneously reconstitute in the absence of CID ligand (19) will further reduce leakage and improve the fidelity of memory. We also treated cells carrying a tandem GPCv2-Fluor with CID ligands in a different temporal order: ABAGA4ABAGA4.…”