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

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 1H alpha CEST pulse sequence which overcomes the resonance overlap problem in the 1H alpha-13C alpha 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 reso-nance coherence transfer pathway from 1H alpha (i) to 1HN(i + 1) during which the 15N(t1) and 1HN(t2) 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 1H alpha 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 1H alpha 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.

Automated summary

Over 40% of eukaryotic proteomes and 15% of bacterial proteomes are predicted to be intrinsically disordered based on their amino acid sequence. NMR spectroscopy has made significant contributions to our understanding of intrinsically disordered proteins (IDPs) due to its ability to characterize heterogeneity at atomic resolution. A new 1H alpha CEST pulse sequence has been developed to overcome resonance overlap problem in the 1H alpha-13C alpha plane of IDPs. This sequence has been applied to determine the excited state 1H alpha chemical shifts of the intrinsically disordered DNA binding domain of the bacterial cytidine repressor.