Creation of Intrinsic Electrochemically-Active Solute Binding Proteins Suitable for Continuous Monitoring Systems
Authors: Hatada Mika , Kane Bryant J. , Takamatsu Shouhei , Okuda-Shimazaki Junko , Tsugawa Wakako , Sode Koji 
Published: December 2024

Introduction

An intrinsic electrochemically-active periplasmic solute binding protein (SBP) was created by genetically fusing an L-Glutamine (L-Gln) binding protein (QBP) with a heme-containing cytochrome as the built-in redox probe. The engineered molecule was characterized for its application in electrochemical continuous biosensing systems.

Periplasmic solute binding proteins (SBPs) recognize and bind their target molecules specifically and reversibly without cofactors or catalytic reactions. A notable characteristic of SBPs is that they change their conformation upon ligand binding. SBPs have been utilized as a recognition element for the development of biosensors by taking the advantage of their conformation change. Indeed, this conformational change can be monitored spectroscopically via SPBs’ intrinsic fluorescence and electrochemically with redox probe modification. We have previously reported the electrochemical continuous monitoring of L-Gln with a QBP modified by a synthetic redox probe, amine-reactive phenazine ethosulfate (arPES) [1]. Here, we utilized square wave voltammetry and intermittent pulse amperometry as the detection methods. The changes in the dynamics of electron transfer between redox probe and the electrode due to the conformation change of QBP upon the binding with L-Gln was monitored electrochemically. However, ideal biosensing molecules would intrinsically contain functions necessary for biosensor construction. For instance, the built-in mediator in enzymes, such as bacterial FAD-dependent glucose dehydrogenase [2] and fructose dehydrogenase [3] allow direct electron transfer. Therefore, we aimed to create an intrinsic electrochemically-active SBP, which harbors a built-in redox probe for applications in electrochemical continuous biosensing systems. In this study, we present the creation and characterization of a QBP-cytochrome fusion as the representative.

Methods

As a representative model, we designed and created a QBP harboring a bacterial cytochrome as the built-in redox probe. For the cytochrome domain, we employed either a c-type or a b-type bacterial cytochrome to investigate their feasibilities as the built-in redox probe, considering their structural and electrochemical characteristics. The bacterial cytochrome c or cytochrome b was genetically fused into a flexible loop region of QBP observed in the crystal structure of Escherichia coli-derived QBP (PDB ID:1GGG), and recombinantly prepared using Escherichia colias the host organism.

Result and discussion

The engineered QBP with heme c containing cytochrome (QBP/cytc), and QBP with heme b containing cytochrome (QBP/cytb),were successfully produced recombinantly as a soluble protein. The binding ability with the ligand was investigated by intrinsic fluorescence measurements and the prepared QBP/cytc or QBP/cytb retained the ability to recognize L-Gln. Furthermore, both QBP/cytc and QBP/cytb showed a characteristic UV/Vis spectrum typical for cytochrome c and b, respectively, showing the presence of heme as the redox center. These results indicated the successful creation of SBPs with a built-in redox probe, eliminating the need for post-harvested chemical modification with a redox probe. The electrochemical characterization of created QBP/cytc and QBP/cytb for the continuouselectrochemical biosensing system of L-Gln will also be presented.

Conclusion

In this study, we demonstrated the creation of an intrinsic electrochemically-active SBP by genetically fusing bacterial cytochrome c or cytochrome b to QBP. Our study showed that the created SBP with a built-in redox probe will be an attractive biosensing molecule inthe development of an SBP-based in vivo or in situ continuous monitoring technology.

References

[1] Takamatsu et al., Sens. Actuators B Chem. 2021, 346, 130554

[2] O-Shimazaki et al., Commun.Biol., 2022, 5, 1334

[3] Suzuki et al., ACS Catal. 2023, 13, 20, 13828–13837