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

Abstract

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 (CytR^N) 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.

Keywords: Alpha proton; Chemical exchange saturation transfer; Conformational change; Intrinsically disordered proteins; Nuclear magnetic resonance spectroscopy; Protein dynamics.

Figure 1. Strategy for acquiring ¹Hα CEST data on IDPs.

A) The ¹Hα region of a constant-time ¹H-¹³C HSQC spectrum of U-¹³C,¹⁵N labelled CytR^N. Green resonances arise from Gly which do not have a scalar coupled sidechain ¹³Cβ. B) ¹H-¹⁵N HSQC spectrum of CytR^N. C) The coherence transfer pathway in the ¹Hα CEST experiment reported here. CEST is carried out on ¹Hα (black dashed square) and the magnetization is then transferred from ¹Hα(i) through ¹³Cα(i) and ¹³CO(i) to ¹⁵N (i+1) and then to ¹HN(i+1) for detection. Red dashed squares indicate nuclei that are frequency labelled in the experiment. D) Cartoon representation of conformational exchange between ground and excited states of a protein where the alpha proton H₁ comes close (< 5 Å) to H₂ in the ground state. E) The spin-state-selective approach for acquiring proton CEST without interference from dipolar cross-relaxation. (Top) ¹H CEST profile of an alpha proton resonating at 4.5 ppm (major dip), exchanging with a second conformation where the chemical shift of the ¹Hα is 5.5 ppm (minor dip). The ¹Hα is proximal to a second ¹Hα which results in an NOE dip at 3.5 ppm. There is no ¹³C-decoupling during the CEST period; therefore doublets separated by ¹J_HαCα are seen for each dip. (Middle) Spin-state-selective CEST data selecting for H_zC_α (blue) and H_zC_β (red). (Bottom) CEST profile of H_zC_α-H_zC_β =2H_zC_z, where the NOE dip cancels out (green).

Figure 2. The pulse sequence for acquiring HACACONNH-based ¹Hα CEST data.

The ¹H carrier is centred on the water resonance except during the CEST period and the ¹⁵N carrier at ~ 118 ppm, while the ¹³C carrier is placed at 58 ppm till gradient g3 and then jumped to 176 ppm for the rest of the pulse sequence. All pulses are applied along the x-axis unless specified otherwise. Narrow and wide rectangles represent 90° and 180° pulses respectively. Open rectangles correspond to rectangular pulses applied 118 ppm off-resonance while the hashed rectangles are rectangular pulses applied -118 ppm off-resonance. ¹H and ¹⁵N pulses are applied at the highest power while ¹³C 90° and 180° pulses are applied at RF field amplitudes of $\xi /\sqrt{15}$ and $\xi /\sqrt{3}$ Hz respectively, where ξ is the frequency difference in Hz between the ¹³Cα and ¹³CO spectral bands (90). ¹H decoupling is implemented via a ~6 kHz WALTZ-16 (91) composite pulse decoupling pattern. Off-resonance ¹³Cα decoupling is carried out with a cosine modulated SEDUCE-1 pattern (92, 93) with a pulse width of 315 μs (at 600 MHz B₀ field strength), and ¹⁵N decoupling during acquisition is carried out with a ~ 1 kHz WALTZ-16 pattern. WURST-2 adiabatic decoupling (94) is used for ¹³Cβ decoupling in the 16.5-41.5 ppm and 68-72 ppm bands. The phase cycling used in the sequence is: ϕ₁ = (-y,y), ϕ₂ = (x,x,-x,-x) and receiver = (x,-x,-x,x). The delays used in the experiment are: τ_b = 1.8 ms, τ_c = 4.4 ms, τ_d = 12.4 ms, τ_e = 5 ms, τ_f = 2.3 ms, T = 12.4 ms, δ = 5.5 ms and Δ = 0.5 ms. The amplitudes and durations of the various gradients in (G/cm, ms) are: g₁: (12000, 0.5), g₂: (-8343, 0.5), g₃: (10000, 0.8), g₄: (30000, 1.25), g₅: (4000, 0.3), g₆: (2000, 0.4), g₇: (30400, 0.125). The two gradients g₄ and g₇ represented as open rectangles are required for coherence selection. Delays χ₁ and χ₂ are set to the larger of the two values (T-t₁/2, 0) and (0, t₁/2-T) respectively, and this allows ¹⁵N chemical shift evolution in t₁ to occur in a semi-constant manner (72, 95). Quadrature detection in the F1 dimension is implemented using enhanced sensitivity gradient coherence selection (–98).

Figure 3. ¹Hα CEST on U-¹³C,¹⁵N labelled ubiquitin.

A) Three-dimensional structure of ubiquitin showing helices, sheets and loops in red, yellow and green respectively (PDB ID: 1UBQ (99)). B) ¹H-¹⁵N HSQC spectrum of ubiquitin. C) ¹Hα CEST profiles of selected residues in ubiquitin that were obtained by analyzing the i+1 correlations indicated in the HSQC spectrum in panel B. ¹Hα CEST data were acquired using the pulse sequence in Figure 2. The ¹Hα CEST profile for Gly10 shows two dips arising from each of the two diastereotopic ¹Hα protons of Gly10.

Figure 4. Characterizing exchange in the folded L24A FF domain using ¹Hα CEST.

A) Cartoon representation of the native (31) and intermediate states of L24A FF (PDB ID:2L9V (31)) in exchange on the millisecond timescale. B) ¹H-¹⁵N HSQC spectrum of L24A FF. Resonances whose ¹Hα CEST profiles are quantified in panel C are labelled in the spectrum. C) ¹Hα CEST profiles of selected resonances of L24A FF. Cyan and red dashed lines indicate the chemical shifts of the native and intermediate states respectively. D) Correlation between the ¹Hα chemical shifts of L24A FF intermediate states acquired using ¹Hα CPMG (y-axis, (31)) and ¹Hα CEST (x-axis). The solid line depicts the y=x function.

Figure 5. Characterizing exchange in the IDP CytR^N using ¹Hα CEST.

A) CytR^N exchanges between a disordered ensemble and a three-helix bundle folded state (52). B) ¹H-¹⁵N HSQC spectrum of disordered CytR^N showing limited spectral dispersion characteristic of an IDP. Resonances for which ¹Hα CEST profiles are quantified in panel C are labelled on the spectrum (except A10, which is not a part of the fragment included in the structure calculation). C) ¹Hα CEST profiles of selected residues of CytR^N. The chemical shift positions of the ground (disordered) and excited (folded) states are indicated by cyan and red dashed lines respectively.

Figure 6. Structure of the CytR^N excited state calculated chemical shifts and RDCs.

A) Composite chemical shift and Rosetta energy score of structures plotted against the RMSD of each structure to the lowest energy structure. B) Overlay of the cartoon representations of the 10 structures with the lowest energy. Comparison between the experimental (red bars) and predicted ¹Hα chemical shifts (yellow circles) for the CytR^N excited state shown as a function of residues (C) and as a correlation plot (D). Predictions were done using Sparta+ on the lowest energy structure. Error bars are uncertainties in residue-specific chemical shift predictions from Sparta+. E) Overlay of the CytR^N excited state calculated without (green) and with (red) ¹Hα chemical shifts. F,G) A comparison of the same experimental data as in panels (C) and (D) with predictions made using Sparta+ (84) on the lowest energy structure calculated without using input ¹Hα chemical shifts (52).