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 19F 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.
Highlights
Nucleic acid-to-protein interactions play an eminent role in living organisms.[1]
Our study focuses on the cold shock protein B from Bacillus subtilis (BsCspB) which three-dimensional structure has been determined by both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.[11]
Fluorine labeled BsCspB (Figure 1) is used to investigate, first, the potential effect of fluorine labeling on protein-to-ligand binding properties by applying fluorescence and NMR spectroscopy under dilute conditions
Summary
Dilute conditions may not precisely report on binding processes taking place in vivo. (ssDNA) with fluorine labeled 4-19F-Phe-BsCspB as well as 5-19FTrp-BsCspB under dilute conditions This experimental setup enables the precise determination of the binding affinity (KD value) and thereby a direct comparison to data regarding binding of ssDNA to wild type BsCspB[13c] is accessible. BsCspB has been fluorine labeled at tryptophan (5-19F-Trp-BsCspB) or phenylalanine positions (4-19F-Phe-BsCspB) depicting key sites for the interaction with oligonucleotides.[16] It has been shown before that neither the structure, stability or the folding kinetics is changed when fluorine modified BsCspB is compared with wild type protein.[17] Note that fluorine labeled BsCspB has been already successfully applied in cell lysate to determine overall thermodynamic stability by using 19F NMR spectroscopy.[18] Here, fluorine labeled BsCspB (Figure 1) is used to investigate, first, the potential effect of fluorine labeling on protein-to-ligand binding properties by applying fluorescence and NMR spectroscopy under dilute conditions. The combination of fluorine labeled proteins and 19F NMR spectroscopy acts for this reason as an excellent tool to probe ligand binding affinity in native surroundings in a quantitative manner
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