Fluorine NMR Spectroscopy Enables to Quantify the Affinity Between DNA and Proteins in Cell Lysate

Abstract

The determination of the binding affinity quantifying the interaction between proteins and nucleic acids is of crucial interest in biological and chemical research. Here, we have made use of site-specific fluorine labeling of the cold shock protein from Bacillus subtilis, BsCspB, enabling to directly monitor the interaction with single stranded DNA molecules in cell lysate. High-resolution 19 F NMR spectroscopy has been applied to exclusively report on resonance signals arising from the protein under study. We have found that this experimental approach advances the reliable determination of the binding affinity between single stranded DNA molecules and its target protein in this complex biological environment by intertwining analyses based on NMR chemical shifts, signal heights, line shapes and simulations. We propose that the developed experimental platform offers a potent approach for the identification of binding affinities characterizing intermolecular interactions in native surroundings covering the nano-to-micromolar range that can be even expanded to in cell applications in future studies.

What remains from living cells in bacterial lysate-based cell-free systems

“…The concentration of cell lysate does not significantly modify the interaction between a ligand and its target protein [68] . However, increasing the concentration of cell lysate within CFS results in a monotonic increase in the thermodynamic stability of the Bacillus subtilis cold shock protein B ( Bs CspB).…”

2′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions

“…However, NMR studies in cellular extracts (bacterial, xenopus oocytes, mammalian) have demonstrated that it is also possible to obtain structural information on macromolecules, such as proteins and nucleic acids, in physiological conditions and they are easier to conduct than in-cell NMR ( Luchinat and Banci, 2022 ; Theillet and Luchinat, 2022 ). For example, NMR studies of nucleic acids in cell extract has been carried out to monitor the maturation of tRNAs in yeast extract ( Barraud et al, 2019 ) or to analyse ssDNA/protein interaction by 19 F labelling of the protein and NMR analysis in E. coli cell lysate ( Welte et al, 2021 ).…”

Solvent Effects Used for Optimal Simultaneous Analysis of Amino Acids via 19F NMR Spectroscopy

“…NMR-active isotope 19 F nuclei with 100% natural abundance exhibit a wide chemical shift range (∼400 ppm), which is exquisitely sensitive to the electronic microenvironment . These merits make 19 F NMR incredibly useful in the fields of analytical chemistry and chemical biology, such as the analysis of a complex multicomponent mixture, − quantification of hydrogen bonds, and the conformational investigation of proteins or nucleic acids. − …”