Solute translocation by membrane transport proteins is a vital biological process that can be tracked, on the sub-second timescale, using nuclear magnetic resonance (NMR). Fluorinated substrate analogues facilitate such studies because of high sensitivity of 19 F NMR and absence of background signals. Accurate extraction of translocation rate constants requires precise quantification of NMR signal intensities. This becomes complicated in the presence of J-couplings, cross-correlations, and nuclear Overhauser effects (NOE) that alter signal integrals through mechanisms unrelated to translocation. Geminal difluorinated motifs introduce strong and hard-to-quantify contributions from non-exchange effects, the nuanced nature of which makes them hard to integrate into data analysis methodologies. With analytical expressions not being available, numerical least squares fitting of theoretical models to 2D spectra emerges as the preferred quantification approach. For large spin systems with simultaneous coherent evolution, cross-relaxation, cross-correlation, conformational exchange, and membrane translocation between compartments with different viscosities, the only available simulation framework is Spinach. In this study, we demonstrate GLUT-1 dependent membrane transport of two model sugars featuring CF2 and CF2 CF2 fluorination motifs, with precise determination of translocation rate constants enabled by numerical fitting of 2D EXSY spectra. For spin systems and kinetic networks of this complexity, this was not previously tractable.
Keywords: GLUT-1; Spinach; exchange spectroscopy; human erythrocyte; nuclear magnetic resonance; polyfluorinated sugar.
© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.