Generalizable Protein Biosensors Based on Synthetic Switch Modules

Abstract

Allosteric protein switches are key controllers of information and energy processing in living organisms and are desirable engineered control tools in synthetic systems. Here we present a generally applicable strategy for construction of allosteric signaling systems with inputs and outputs of choice. We demonstrate conversion of constitutively active enzymes into peptide-operated synthetic allosteric ON switches by insertion of a calmodulin domain into rationally selected sites. Switches based on EGFP, glucose dehydrogenase, NanoLuciferase, and dehydrofolate reductase required minimal optimization and demonstrated a dynamic response ranging from 1.8-fold in the former case to over 200-fold in the latter case. The peptidic nature of the calmodulin ligand enables incorporation of such synthetic switch modules into higher order sensory architectures. Here, a ligand-mediated increase in proximity of the allosteric switch and the engineered activator peptide modulates biosensor’s activity. Created biosensors were used to measure concentrations of clinically relevant drugs and biomarkers in plasma, saliva, and urine with accuracy comparable to that of the currently used clinical diagnostic assays. The approach presented is generalizable as it allows rapid construction of efficient protein switches that convert binding of a broad range of analytes into a biochemical activity of choice enabling construction of artificial signaling and metabolic circuits of potentially unlimited complexity.

Boolean Logic Networks Mimicked with Chimeric Enzymes Activated/Inhibited by Several Input Signals

“…The CaM receptor unit changes its conformation dramatically upon binding Ca 2+ cations and M13 peptide cooperatively, Figure A. This conformation change transfers to the PQQ‐GDH enzyme backbone affecting the biocatalytic activity . The PQQ‐GDH‐CaM chimeric enzyme is catalytically inactive when the CaM receptor unit exists in the extended state in the absence of M13 peptide, Figure A.…”

“…The second multi‐input logic system was realized with another chimeric enzyme, PQQ‐GDH fused with the affinity clamp, Figure A. While conceptually preparation of the PQQ‐GDH‐Clamp enzyme was similar to the PQQ‐GDH‐CaM, the function of the clamp was the opposite .…”

“…The second multi‐input logic system was realized with another chimeric enzyme, PQQ‐GDH fused with the affinity clamp, Figure A. While conceptually preparation of the PQQ‐GDH‐Clamp enzyme was similar to the PQQ‐GDH‐CaM, the function of the clamp was the opposite . The enzyme was active when the clamp was in the “open” conformation, but binding of its cognate peptide and formation of the “closed” clamp conformation resulted in distortion of the enzyme backbone structure and inhibition of its reaction, Figure A.…”

“…The input signals processed by the fused receptors can be physiologically important biomarkers (e. g., α‐amylase, cyclosporine, tacrolimus, human serum albumin, etc. ), which represent particular interest for biomedical applications. The logic schemes can be designed according to the information processing needed, being much more sophisticated than single Boolean gates.…”

“…Preparation of the chimeric PQQ‐GDH‐CaM and PQQ‐GDH‐Clamp enzymes, composition of the binding peptides and experimental details on the enzyme assay reactions were published elsewhere . KRRWKKNFIAVSAANRFKKISSSGAL (CaM‐binding peptide) and RGSIDTWV (the clamp‐binding peptide) were purchased from AnaSpec, Fremont, CA, USA.…”