Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction

Abstract

Biosensors for signaling molecules allow the study of physiological processes by bringing together the fields of protein engineering, fluorescence imaging, and cell biology. Construction of genetically encoded biosensors generally relies on the availability of a binding "core" that is both specific and stable, which can then be combined with fluorescent molecules to create a sensor. However, binding proteins with the desired properties are often not available in nature and substantial improvement to sensors can be required, particularly with regard to their durability. Ancestral protein reconstruction is a powerful protein-engineering tool able to generate highly stable and functional proteins. In this work, we sought to establish the utility of ancestral protein reconstruction to biosensor development, beginning with the construction of an L-arginine biosensor. L-arginine, as the immediate precursor to nitric oxide, is an important molecule in many physiological contexts including brain function. Using a combination of ancestral reconstruction and circular permutation, we constructed a F€ orster resonance energy transfer (FRET) biosensor for L-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity, with a K d of~14 mM and a maximal dynamic range of 35%. Importantly, cpFLIPR was highly robust, enabling accurate L-arginine measurement at physiological temperatures. We established that cpFLIPR is compatible with two-photon excitation fluorescence microscopy and report L-arginine concentrations in brain tissue.

The interplay of protein–ligand and water‐mediated interactions shape affinity and selectivity in the LAO binding protein

“…For this reason, such proteins have been good models to study the structural basis that determine affinity and selectivity in proteins [6,[15][16][17][18][19][20][21][22][23][24][25]. These features have made PBPs a suitable target to develop biosensors [14,[26][27][28][29][30][31] and design new binding abilities [14,32,33].…”

The thermostability and specificity of ancient proteins

“…This is of deep interest to evolutionary biochemists seeking to identify the general principles that shape protein evolution. Further, a trend could mean that sampling evolutionary history would provide access to qualitatively different proteins [13••] — a boon to engineers looking for proteins with desirable properties as templates for further engineering [14,15]. …”

Identification of novel inhibitors of the amino acid transporter B0AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

“…Here, we established the FLIPR system as a suitable assay to screen large compound libraries and to characterize amino acid transport inhibitors. Alternatively, FRET sensors can be used to detect amino acid transport (Whitfield et al, 2015;Vanoaica et al, 2016). However, these monitor amino acid accumulation through any transport system and therefore require secondary screening to ensure targeting is correct.…”