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 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(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 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.

Functional regulation of an intrinsically disordered protein via a conformationally excited state.

A longstanding goal in the field of intrinsically disordered proteins (IDPs) is to characterize their structural heterogeneity and pinpoint the role of this heterogeneity in IDP function. Here, we use multinuclear chemical exchange saturation (CEST) nuclear magnetic resonance to determine the structure of a thermally accessible globally folded excited state in equilibrium with the intrinsically disordered native ensemble of a bacterial transcriptional regulator CytR. We further provide evidence from double resonance CEST experiments that the excited state, which structurally resembles the DNA-bound form of cytidine repressor (CytR), recognizes DNA by means of a "folding-before-binding" conformational selection pathway. The disorder-to-order regulatory switch in DNA recognition by natively disordered CytR therefore operates through a dynamical variant of the lock-and-key mechanism where the structurally complementary conformation is transiently accessed via thermal fluctuations.

Quantification of Entropic Excluded Volume Effects Driving Crowding-Induced Collapse and Folding of a Disordered Protein.

We investigate the conformational properties of the intrinsically disordered DNA-binding domain of CytR in the presence of the polymeric crowder polyethylene glycol (PEG). Integrating circular dichroism, nuclear magnetic resonance, and single-molecule Förster resonance energy transfer measurements, we demonstrate that disordered CytR populates a well-folded minor conformation in its native ensemble, while the unfolded ensemble collapses and folds with an increase in crowder density independent of the crowder size. Employing a statistical-mechanical model, the effective reduction in the accessible conformational space of a residue in the unfolded state is estimated to be 10% at 300 mg/mL PEG8000, relative to dilute conditions. The experimentally consistent PEG-temperature phase diagram thus constructed reveals that entropic effects can stabilize disordered CytR by 10 kJ mol-1, driving the equilibrium toward folded conformations under physiological conditions. Our work highlights the malleable conformational landscape of CytR, the presence of a folded conformation in the disordered ensemble, and proposes a scaling relation for quantifying excluded volume effects on protein stability.

Metamorphic proteins: the Janus proteins of structural biology.

The structural paradigm that the sequence of a protein encodes for a unique three-dimensional native fold does not acknowledge the intrinsic plasticity encapsulated in conformational free energy landscapes. Metamorphic proteins are a recently discovered class of biomolecules that illustrate this plasticity by folding into at least two distinct native state structures of comparable stability in the absence of ligands or cofactors to facilitate fold-switching. The expanding list of metamorphic proteins clearly shows that these proteins are not mere aberrations in protein evolution, but may have actually been a consequence of distinctive patterns in selection pressure such as those found in virus-host co-evolution. In this review, we describe the structure-function relationships observed in well-studied metamorphic protein systems, with specific focus on how functional residues are sequestered or exposed in the two folds of the protein. We also discuss the implications of metamorphosis for protein evolution and the efforts that are underway to predict metamorphic systems from sequence properties alone.