Extending the Sensitivity of CEST NMR Spectroscopy to Micro-to-Millisecond Dynamics in Nucleic Acids Using High-Power Radio-Frequency Fields
Biomolecules undergo motions on the micro-to-millisecond timescale to adopt low-populated transient states that play important roles in folding, recognition, and catalysis. NMR techniques such as CPMG, CEST and R1\xcf\x81 are the most commonly used methods for characterizing such transitions at atomic resolution under solution conditions. CPMG and CEST are most effective at characterizing motions on the millisecond timescale. While some implementations of the R1\xcf\x81 experiment are more broadly sensitive to motions on the micro-to-millisecond timescale, they entail the use of selective irradiation schemes and inefficient 1D data acquisition methods. Here, we show that high power radio-frequency fields can be used in CEST experiments to extend sensitivity to faster motions on the micro-to-millisecond timescale. Given the ease of implementing high power fields in CEST, the work described here should make it easier to characterize micro-to-millisecond dynamics in biomolecules.
<p>Proteins and nucleic acids undergo structural dynamics over timescales ranging from picoseconds to seconds.[1] Motions on the micro-to-millisecond timescale frequently involve transitions from the energetically most favorable ground state (GS) confoWe first examined exchange between rmation towards low-populated (typically < 5%) and short-lived (lifetime < 2 ms) excited conformational states (ESs) that typically have non-native conformations.[2] There has been great interest in characterizing such motions because they have been shown to play critical roles in folding[3] and function,[4] and because many ESs are attractive drug targets.[5] Different NMR experiments have been developed to characterize micro-to-millisecond timescale motions that rely on modulating the dephasing of magnetization due to chemical exchange via the application of radio-frequency (RF) fields.[6] Spin-relaxation in the rotating frame (R1ρ)[7] is most effective at characterizing fast microsecond timescale motions while Carr-Purcell-Meiboom-Gill (CPMG)[8] and Chemical Exchange Saturation Transfer (CEST)[9] are most effective at characterizing slower millisecond motions. Here, we show that use of high power RF fields extends the sensitivity of CEST to faster timescale motions. Given other established technical advantages of CEST,[10] this broadens the scope of characterizing micro-to-millisecond motions in biomolecules using NMR.</p><p>An exchange process is most effectively characterized when the effective field (ωeff, in rad/s) experienced by a given nucleus is comparable to the rate of exchange (kex = k1 + k−1, where k1 and k−1 are the forward and backward rate constants, respectively).[11] Under these conditions, dephasing of magnetization due to chemical exchange is maximally modulated by the applied RF field. CPMG, CEST, and R1ρ have different timescale sensitivities because they employ different schemes to apply the RF field, which in turn place different constraints on the range of ωeff values that can be attained.</p><p>CPMG employs a series of hard 180° pulses with varying time intervals to modulate dephasing of transverse magnetization.[12] The lower limit of ωeff/2π ~ 300 Hz is determined by signal losses due to relaxation, while the upper limit of ωeff/2π ~ 12 kHz is determined by the probe tolerance to high power RF fields and sample heating.[10b, 11] Thus, CPMG is typically sensitive to kex between ~200 s−1 and ~4,000 s−1.[12] Higher ωeff (~100 Hz < ωeff/2π < ~18kHz) are accessible in the R1ρ experiment, which employs spin-locking RF fields with variable power (~100 Hz < ω1/2π < ~5 kHz) and offset[13]. Therefore, R1π is typically sensitive to a wider range of kex (~400 s−1 < kex < ~50,000 s−1) with the lower limit being determined by complications due to the evolution of homonuclear scalar couplings when using weak spin-locking fields,[10c] and the upper limit by the tolerance of the probe to high power RF fields.[14] Optimal sensitivity to slower exchange rates however typically necessitates the use of selective excitation for interrogating one spin at a time using 1D data acquisition schemes.[15] In contrast to R1ρ, both CPMG and CEST are routinely performed in a 2D manner. CEST involves monitoring dephasing of longitudinal magnetization as a function of the applied RF field strength and offset frequency.[10a, 10b, 16] Weaker RF fields (ω1/2π < 100 Hz) relative to R1ρ can also be used in CEST, making it more effective at characterizing slower exchange.[10a, 17] CEST studies of biomolecules have typically used low power RF fields (ω1/2π < 100 Hz) as higher powers lead to broadening of the dips in the CEST profiles, making it difficult to resolve distinct chemical shifts for the GS and ES.[16] This has limited the applicability of CEST to processes in the slow exchange regime with kex between ~20 s−1 and ~400 s−1.[10a]</p><p>Given the advantages of CEST relative to R1ρ,[10a] including its ease of implementation, we examined whether we could extend the sensitivity of CEST to faster timescale motions by employing higher power RF fields. In theory, there are no technical restrictions on using RF fields with powers as high as those used in R1ρ.[18] Furthermore, studies have successfully extracted exchange parameters from CEST profiles of small molecule systems in the intermediate-fast exchange regime using low[19] and high[18a, 18b] power RF fields even though distinct dips for the GS and ES were not observed (Discussion S1).</p><p>To test whether it is indeed feasible to characterize faster exchange processes using high power CEST, we used the Bloch-McConnell (B-M) equations to simulate CEST and R1ρ profiles for two-state GS↔ES exchange as a function of varying RF powers and kex. For small kex (300 s−1, τex = 1/kex = 3.3 ms) in the slow exchange regime (kex/Δω < 1), conventional low power CEST profiles displayed the characteristic dip at an offset corresponding to the ES chemical shift (ωES) (Figure 1). As kex was increased to the intermediate (kex = 3,000 S−1, τex = 0.33 ms, kex/Δω ~ 1) and fast (kex = 9,000 s−1, τex = 0.11 ms, kex/Δω > 1) exchange regimes, the ES dip gradually broadened and diminished in size, and the profiles became increasingly insensitive to the exchange process, showing little deviations from the profiles simulated in the absence of exchange (Figure 1, solid vs. dashed lines).[10a] This highlights the challenge of characterizing faster processes using low power CEST. As expected, for small kex in the slow exchange regime, the ES dip was broadened out to a shoulder with increasing RF powers that was more pronounced for offsets in the direction of ωES relative to offsets in the direction opposite to ωES. This behaviour is analogous to that seen routinely in off-resonance R1ρ.[20] Interestingly, the shoulder in the high power CEST profiles persisted even for large kex values in the intermediate and fast exchange regimes, when the low power CEST profiles were increasingly insensitive to the exchange (Figure 1). Furthermore, the size of the shoulder qualitatively mirrored the magnitude of Rex in the high spin-lock R1ρ profiles. Additional simulations showed that the high power CEST profiles could be fit to the B-M equations to accurately extract the exchange parameters (Figure S1). This indicates that using high power RF fields in CEST could broaden the accessible window to faster exchange rates.</p><p>We tested the feasibility of using high power CEST to characterize two exchange processes on the slow-to-intermediate exchange timescale with kex ~ 3,000 s−1. We first examined exchange between wobble G-T mismatches and their Watson-Crick like counterparts formed by tautomerization of the bases (Figure 2A). This exchange process was previously characterized using off-resonance R1ρ experiments[21] targeting the base imino nitrogen (G-N1, T-N3) and carbon (T-C6) nuclei (Figure 2B, Tables S1-S2 and Discussion S2). The magnitude of kex = 2689 ± 163 s−1 exceeds the typical detection limit of ~400 s−1 for low power CEST.[10a] Consequently, as expected, low power CEST profiles for these nuclei did not show ES dips and exhibited minor deviations from simulated profiles in the absence of exchange (Figure 2B, dashed lines). Moreover, fitting of the profiles did not yield reliable exchange parameters (Figures S2-S3, Tables S1-S2, S4). In contrast, as predicted from B-M simulations (Figure 1), the high power CEST profiles exhibited ES dips (G15-N1 and T5-N3, owing to a large Δω) or a broad shoulder (T5-C6, owing to a smaller Δω) (Figure 2B). This shoulder is best visualized when comparing the normalized intensity for a given positive value of the offset Ωi relative to its corresponding negative counterpart −Ωi (Figure 2C), in which intensities are smaller for offsets in the direction of ωES. Fitting of the high power CEST profiles was significantly improved with the inclusion of conformational exchange and RF field inhomogeneity (Figures S4-S6, Table S3, Materials and Methods), which was essential to obtain accurate estimates of the exchange parameters. In addition, the influence of homonuclear 13C-13C scalar couplings (1JC5-C6 ~ 67 Hz) when fitting the CEST data for T5-C6 was considered only for the low RF power profiles for which ω1/2π < 1JC-C, as they are suppressed when using high RF powers for which ω1/2π > 1JC-C[10a] (Materials and Methods). The profiles for G15-N1, T5-N3 and T5-C6 could be globally fitted to yield ES population pB = 0.180 ± 0.001%, kex = 3025 ± 19 s−1 (τex = 0.33 ms) and chemical shift differences between the ES and GS (Δω = ωES - ωGS) that were in excellent agreement with those obtained using R1ρ (Figures 2D, S3, Tables S1-S2, S4, Discussions S2-S3). In addition, as negative controls, nuclei with flat R1ρ profiles with no dispersion (G15-C1' and T5-C1') (Figure 3) also showed no signs of chemical exchange in high power CEST. The normalized intensities for positive and negative offsets for these nuclei were similar to each other (Figure 3), and their profiles showed no visual improvement in fit quality on inclusion of exchange (Figure S7).</p><p>As a second test, we used high power CEST to characterize the exchange between A-T Watson-Crick and Hoogsteen base pairs that are formed by flipping the adenine base about the glycosidic bond. These experiments were performed on a uniformly 13C/15N labelled DNA duplex (A6-DNA, Figure 4A). Based on R1ρ, this process can be characterized using the adenine-C8 and sugar adenine-C1' nuclei (Figure 4B, Table S5). As expected given the large kex = 3775 ± 163 s−1, low power CEST profiles for these nuclei did not display ES dips, showed minor deviations from simulated profiles in the absence of exchange (Figure 4B, dashed lines), and did not yield reliable exchange parameters on fitting (Figures S3, S8, Table S5). Furthermore, the exchange was more effectively characterized using high power RF fields, which yielded CEST profiles for A16-C1' and A16-C8 with broad shoulders (Figures 4B–C) that could be globally fitted to yield pB = 0.316 ± 0.009 %, kex = 3585 ± 70 s−1 (τex = 0.28 ms) and ES Δω values that were in excellent agreement with those determined using R1ρ (Figures 4D, S3, Table S5, Discussion S3). Again, nuclei with flat R1ρ profiles with no dispersion (A16-C2, T9-C6 and T9-C1') did not show detectable exchange in high power CEST experiments (Figures 4B–C, S9).</p><p>In conclusion, high power RF fields extend the window of exchange timescales accessible to CEST, from milliseconds in the slow exchange regime, to micro-to-milliseconds in the intermediate and fast exchange regimes (Discussion S4). By aligning the magnetization of all nucleic along z, CEST effectively overcomes limitations related to aligning magnetization of many nuclei that have limited the applicability of 2D R1ρ experiments.[23] By making it easier to characterize biomolecular dynamics on the micro-to-millisecond timescale, we anticipate that the approach outlined in this study will facilitate the characterization of ESs in nucleic acids and proteins.</p>