Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant

Abstract

<b><i>Aims:</i></b> Genetically encoded green fluorescent protein (GFP)-based redox biosensors are widely used to monitor specific and dynamic redox processes in living cells. Over the last few years, various biosensors for a variety of applications were engineered and enhanced to match the organism and cellular environments, which should be investigated. In this context, the unicellular intraerythrocytic parasite <i>Plasmodium</i>, the causative agent of malaria, represents a challenge, as the small size of the organism results in weak fluorescence signals that complicate precise measurements, especially for cell compartment-specific observations. To address this, we have functionally and structurally characterized an enhanced redox biosensor superfolder roGFP2 (sfroGFP2). <b><i>Results:</i></b> SfroGFP2 retains roGFP2-like behavior, yet with improved fluorescence intensity (FI) <i>in cellulo</i>. SfroGFP2-based redox biosensors are pH insensitive in a physiological pH range and show midpoint potentials comparable with roGFP2-based redox biosensors. Using crystallography and rigidity theory, we identified the superfolding mutations as being responsible for improved structural stability of the biosensor in a redox-sensitive environment, thus explaining the improved FI <i>in cellulo</i>. <b><i>Innovation:</i></b> This work provides insight into the structure and function of GFP-based redox biosensors. It describes an improved redox biosensor (sfroGFP2) suitable for measuring oxidizing effects within small cells where applicability of other redox sensor variants is limited. <b><i>Conclusion:</i></b> Improved structural stability of sfroGFP2 gives rise to increased FI <i>in cellulo</i>. Fusion to hGrx1 (human glutaredoxin-1) provides the hitherto most suitable biosensor for measuring oxidizing effects in <i>Plasmodium</i>. This sensor is of major interest for studying glutathione redox changes in small cells, as well as subcellular compartments in general. <i>Antioxid. Redox Signal.</i> 37, 1-18.