Fig. 3: Design and optimization of the LHGFR.
From: An l-2-hydroxyglutarate biosensor based on specific transcriptional regulator LhgR
a Schematic representation of the predicted conformational change of LhgR-based l-2-HG biosensor LHGFR in the absence or presence of l-2-HG. In particular, schematic representations of the tertiary structure of Venus and mTFP shown were predicted based on the respective protein sequences. b Dose-response curve of purified LHGFR0N0C for increasing concentrations (10 nM to 1 mM) of l-2-HG in 50 mM Tris-HCl buffer (pH 7.4). The emission ratio of Venus to mTFP increased (430 nm excitation) after l-2-HG binding. c Heap map of the truncating the N-terminal and C-terminal amino acids of LhgR to ∆Rmax. The color indicates the value of ∆Rmax and white indicates the untested variants. d Comparison of the ∆Rmax of a set of l-2-HG biosensor variants based on the C-terminal amino acid truncated of LhgR. e, f Specificities of the purified LHGFR0N3C (e) and LHGFR0N7C (f). The emission ratio changes of both biosensors were measured in the presence of 240 μM d-lactate, l-lactate, l-2-HG, d-2-HG, or different intermediates of the TCA cycle and l-lysine catabolism. g, h Reversal of l-2-HG binding with LHGFR by conversion of l-2-HG to 2-KG. The emission ratio of purified LHGFR0N3C (g) and LHGFR0N7C (h) was recorded in the absence and presence of 20 μM l-2-HG before and after the addition of 5 μM purified LhgO for 25 min. All data shown are means ± s.d. (n = 3 independent experiments).
