Probing excited state 1Hα chemical shifts in intrinsically disordered proteins with a triple resonance-based CEST experiment: Application to a disorder-to-order switch.

• Characterizing the heterogeneity in intrinsically disordered protein ensembles is vital for understanding their function. • The NMR of IDPs is challenging because of poor spectral dispersion and severe resonance overlap. • We integrate spin-state-selective CEST and triple resonance coherence transfer to design a pulse sequence for measuring 1Hα excited state chemical shifts. • The pulse sequence is used for measuring excited state shifts in the intrinsically disordered DNA binding domain of the cytidine repressor (CytRN) • The structure of the CytRN excited state calculated from the 1Hα chemical shifts is a three-helix bundle with a helix-turn-helix motif. Over 40% of eukaryotic proteomes and 15% of bacterial proteomes are predicted to be intrinsically disordered based on their amino acid sequence. Intrinsically disordered proteins (IDPs) exist as heterogeneous ensembles of interconverting conformations and pose a challenge to the structure–function paradigm by apparently functioning without possessing stable structural elements. IDPs play a prominent role in biological processes involving extensive intermolecular interaction networks and their inherently dynamic nature facilitates their promiscuous interaction with multiple structurally diverse partner molecules. NMR spectroscopy has made pivotal contributions to our understanding of IDPs because of its unique ability to characterize heterogeneity at atomic resolution. NMR methods such as Chemical Exchange Saturation Transfer (CEST) and relaxation dispersion have enabled the detection of 'invisible' excited states in biomolecules which are transiently and sparsely populated, yet central for function. Here, we develop a 1Hα CEST pulse sequence which overcomes the resonance overlap problem in the 1Hα-13Cα plane of IDPs by taking advantage of the superior resolution in the 1H-15N correlation spectrum. In this sequence, magnetization is transferred after 1H CEST using a triple resonance coherence transfer pathway from 1Hα (i) to 1HN(i + 1) during which the 15N(t 1) and 1HN(t 2) are frequency labelled. This approach is integrated with spin state-selective CEST for eliminating spurious dips in CEST profiles resulting from dipolar cross-relaxation. We apply this sequence to determine the excited state 1Hα chemical shifts of the intrinsically disordered DNA binding domain (CytRN) of the bacterial cytidine repressor (CytR), which transiently acquires a functional globally folded conformation. The structure of the excited state, calculated using 1Hα chemical shifts in conjunction with other excited state NMR restraints, is a three-helix bundle incorporating a helix-turn-helix motif that is vital for binding DNA. [ABSTRACT FROM AUTHOR]