¹⁹F NMR Spectroscopy Tagging and Paramagnetic Relaxation Enhancement‐Based Conformation Analysis of Intrinsically Disordered Protein Complexes

Abstract

The combination of 19F NMR spectroscopy tagging and paramagnetic relaxation enhancement (PRE) NMR spectroscopy experiments was evaluated as a versatile method to probe protein–protein interactions and conformational changes of intrinsically disordered proteins upon complex formation. The feasibility of the approach is illustrated with an application to the Myc‐Max protein complex; this is an oncogenic transcription factor that binds enhancer box DNA fragments. The single cysteine residue of Myc was tagged with highly fluorinated [19F]3,5‐bis(trifluoromethyl)benzyl bromide. Structural dynamics of the protein complex were monitored through intermolecular PREs between 19F‐Myc and paramagnetic (1‐oxyl‐2,2,5,5‐tetramethyl‐Δ3‐pyrroline‐3‐methyl)methanethiosulfonate (MTSL)‐tagged) Max. The 19F R2 relaxation rates obtained with three differently MTSL‐tagged Max mutants revealed novel insights into the differential structural dynamics of Myc‐Max bound to DNA and the tumour suppressor breast cancer antigen 1. Given its ease of implementation, fruitful applications of this strategy to structural biology and inhibitor screening can be envisaged.

19F NMR as a tool in chemical biology

“…Moreover, results from these studies revealed that the fibril-forming path of α-Syn might occur without accumulation of soluble low molecular weight intermediates, as no new 19 F NMR signals were observed during the time-course of the aggregation process. 19 F NMR spectroscopy has also now been employed to analyse the conformational dynamics of IDPs upon tertiary and quaternary complex formation [79]. The Myc-Max heterodimer is a well-known oncogenic transcription factor complex able to bind to enhancer box (E-box) regions (5'-CACGTG-3') of DNA with low-nanomolar affinity, what triggers its biological function as a transcriptional regulator [80].…”

19F NMR viewed through two different lenses: ligand-observed and protein-observed19F NMR applications for fragment-based drug discovery

“…PRE measurements have also been used to show dynamic conformational effects within Myc upon Myc-Max heterodimer formation and interactions with DNA. 147 In-cell 19 F NMR The absence of 19 F in biological systems makes the transition to in-cell studies a natural advancement, as it avoids spectral background signals. Early studies were completed in the yeast S. cerevisiae by inducible expression of target proteins and labeling with 5FW to measure protein mobility in cells.…”

“…PRE measurements have also been used to show dynamic conformational effects within Myc upon Myc-Max heterodimer formation and interactions with DNA. 147 …”

How to assess the structural dynamics of transcription factors by integrating sparse NMR and EPR constraints with molecular dynamics simulations

“…Recently, approaches based on the site-specific introduction of paramagnetic labels could characterize the DNA-binding sites of (partially) disordered TFs [61] , [62] , [63] , [64] , [65] . These approaches employed the so-called paramagnetic relaxation enhancement (PRE) technique [66] .…”