Fig. 2: Changes in fluorescence emission spectra in response to glucose binding.
The selected conjugates represent three main apparent color switching patterns upon binding glucose (Glc). Apparent colors were classified according to the spectral barychrome34, λB, value: Blue < 500 nm ; 500 nm < Green < 520 nm: Yellow >520 nm. a Green→Blue, ttGBP.17C; spectral analysis for ttGBP.17C•Acrylodan is shown in detail (note that the ttGBP.17C•Badan exhibits a larger change). b Green→Blue, ecGBP.16C; analysis for Badan conjugate is detailed. c Blue→Green, ttGBP.182C; Acrylodan conjugate is detailed (ttGBP.182C•Badan conjugate is non-responsive). d Green→Yellow, ecGBP.183C; Badan conjugate is detailed (response of ecGBP.183C•Acrylodan conjugate is similar to ttGBP.182C•Acrylodan). Column 1, emission spectra (conjugated fluorophore is indicated; arrows: direction of intensity changes upon glucose addition; cyan: apo-protein spectrum; blue, spectrum at [Glc]≈5xKd; magenta: spectrum at maximal [Glc]). Column 2, apparent color switching monitored by λB. Solid lines: binding isotherm fit using linear baselines for the glucose complex; circles: experimental data. Column 3, change in the logarithm of total emission intensity relative to the apo-protein spectrum, ρ (ρ=0 in the absence of glucose; positive, intensity increases with glucose addition; negative intensity decreases); solid lines: binding isotherm fit with linear baselines for the glucose complex; circles: experimental data; the variations in the fit models (± one standard deviation), calculated from 10,000 bootstrap trials, are shown as vertical gray lines at each experimental data point (distributions may be smaller than the data point circles). Column 4, top three spectral components of the SVD of emission spectra glucose dependence (color-coded numbers indicate peak or shoulder wavelengths; positive peak, increases with glucose; negative peak, decreases with glucose): black, C1, largely invariant; red, C2, the most glucose-responsive spectral component (peaks of opposite signs mark electronic transitions that exchange in response to glucose binding); gray, C3, the component primarily encoding the solvatochromic response at high glucose concentrations. Column 5, binding isotherm fits of the glucose-dependent contributions of the top three components, using a linear baseline for the glucose complex; the three isotherms have identical Kd values. For each component, 1σ variations are shown as gray vertical lines. Additional spectra and SVD decompositions are shown in Supplementary Figs 1–4.
