Quantitative Analysis of 2D EXSY NMR Spectra of Strongly Coupled Spin Systems in Transmembrane Exchange

• Published in: Chembiochem : a European journal of chemical biology 2024-02, Vol.25 (3), p.e202300597-n/a
• Main Authors: Shishmarev, Dmitry, Fontenelle, Clement Q., Linclau, Bruno, Kuprov, Ilya, Kuchel, Philip W.
• Format: Article
• Language: English
• Subjects: Biological activity; Computer Simulation; Couplings; Data analysis; exchange spectroscopy; Exchanging; Fluorination; GLUT-1; human erythrocyte; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy - methods; Mathematical analysis; Mathematical models; Membranes; Molecular Conformation; NMR; Nuclear magnetic resonance; polyfluorinated sugar; Protein transport; Rate constants; Spectra; Spinach; Substrates; Translocation; Two dimensional analysis
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202300597

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 19F 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 CF2CF2 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. Quantitative interpretation of cross‐peaks in 2D EXSY spectra of compounds with strongly coupled spins undergoing transmembrane exchange required brute‐force numerical fitting to extract rate constants.