A single mutation is sufficient to alter ligand binding specificity in a trehalose/maltose genetically encoded fluorescent biosensor

Tre-C04, a green fluorescent protein (GFP)-derived biosensor, detects trehalose and maltose at nanomolar levels by integrating a trehalose/maltose-binding protein from Thermococcus litoralis with a circularly permuted GFP. Ligand binding induces fluorescence, which enables disaccharide quantification. However, its dual specificity limits its utility in applications that require maltose specificity. This study aimed to engineer a Tre-C04 variant with enhanced maltose specificity. Structural analysis and docking simulations identified key residues involved in trehalose and maltose binding. Alanine scanning mutagenesis pinpointed glutamic acid (E17) and arginine (R364) significantly reduced trehalose binding while retaining maltose recognition. Substitutions, including E17D, were tested, yielding a variant named “Malefficient” which exhibited high maltose specificity. Malefficent exhibited a dynamic detection range of 3 nM to 7 µM and a maltose dissociation constant of 295.1 nM. It showed no fluorescence response to other sugars or maltose-derived oligosaccharides, confirming its specificity. The fluorescence signal peaked within five minutes, providing a rapid and sensitive detection system. The engineered biosensor selectively detects maltose with nanomolar sensitivity and rapid response time, making it a promising tool for food processing, beverage quality control, and medical diagnostics. Its ability to function in raw samples without purification enhances its practicality. Future advancements in protein engineering could expand its detection range and accuracy, broadening its application in diverse fields.