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Characterizing Fast Conformational Exchange of Aromatic Rings Using Residual Dipolar Couplings: Distinguishing Jumplike Flips from Other Exchange Mechanisms

Aromatic ring flips are a hallmark of protein dynamics. They are experimentally studied by NMR spectroscopy, where recent advances have led to improved characterization across a wide range of time scales. Results on different proteins have been interpreted as continuous diffusive ring rotations or jumplike flips, leading to diverging views of the protein interior as being fluidlike or solidlike, respectively. It is challenging to distinguish between these mechanisms and other types of conformational exchange because chemical-shift-mediated line broadening provides only conclusive evidence for ring flips only if the system can be moved from the slow- to intermediate/fast-exchange regime. Moreover, whenever the chemical shift difference between the two symmetry-related sites is close to zero, it is not generally possible to determine the exchange time scale. Here we resolve these issues by measuring residual dipolar coupling (RDC)-mediated exchange contributions using NMR relaxation dispersion experiments on proteins dissolved in dilute liquid crystalline media. Excellent agreement is found between the experimental difference in RDC between the two symmetry-related sites and the value calculated from high-resolution X-ray structures, demonstrating that dynamics measured for F52 in the B1 domain of protein G reports on distinct, jumplike flips rather than other types of conformational exchange.


■ INTRODUCTION
Conformational dynamics of proteins is essential for biological function. Aromatic ring flips, i.e., 180°rotations of the χ 2 dihedral angle in Phe and Tyr side chains, are a hallmark of transient conformational fluctuations in proteins. 1,2 Aromatic side chains in the interior of globular proteins commonly undergo rapid rotations, as gauged by the observation of a single peak in the NMR spectrum originating from the two symmetry related nuclei of the ring. In the past, ring flip rates have been measured for only a limited number of proteins by using line shape analysis or longitudinal exchange experiments. 1,3−6 The development of site-selective isotope labeling schemes for aromatic side chains 7−14 has enabled relaxation dispersion experiments for aromatic 13 C and 1 H sites 15−18 and led to a renaissance in studying ring flips by NMR spectroscopy. 19−25 The rotation of an aromatic side chain located inside the protein core requires that the surrounding side chains transiently create sufficient volume to accommodate the ring rotation. Temperature-and pressure-dependent experiments make it possible to determine the activation enthalpy (ΔH ‡ ), entropy (ΔS ‡ ), and volume (ΔV ‡ ) of this process. 1,5,20,21,26,27 Recently, we presented a combined analysis of temperatureand pressure-dependent ring flip data that enabled us to also determine the change in compressibility (Δκ ‡ ) between ground and transition states of a ring flip, 19 which indicates that the transition state is liquidlike, whereas the ground state is solidlike. This result differs from conclusions reached in another study of the dynamics of surface-exposed aromatic residues in ubiquitin, which suggest that the ground state is liquidlike and the aromatic rings undergo continuous diffusive rotations. 24 Thus, further studies are warranted to provide detailed insights into the mechanism and energetics of aromatic ring dynamics and how these properties might differ among aromatic side chains located in different environments, e.g., in the protein core or closer to the protein surface.
To date, the study of ring flips by solution-sate NMR spectroscopy has mainly relied on measuring exchange contributions to the line width (or longitudinal exchange for very slow cases), caused by differences in the chemical shifts of the symmetry-related pairs of 1 Hδ, 1 Hε, 13 Cδ, or 13 Cε nuclei. However, it is not uncommon that none of these sites display significant differences in chemical shift, thereby making ring flip studies by NMR relaxation difficult or impossible. 20,22,28 Furthermore, when separate peaks cannot be observed for the two sides of the aromatic ring, it has been impossible to determine with certainty that exchange broadening is due to ring flips rather than other types of conformational exchange.
Here we demonstrate that these impediments can be resolved by studying exchange broadening mediated by residual dipolar couplings, 29,30 introduced by partial alignment of the protein in dilute liquid crystalline media. 31 The use of RDC-mediated conformational exchange is particularly powerful in the case of aromatic ring flips because the approach resolves cases where the chemical shift difference is (close to) zero and, importantly, provides critical structural information to distinguish whether aromatic rings undergo distinct, jumplike flips, or continuous diffusive ring rotation as invoked recently, 24 or other types of conformational exchange.

■ MATERIALS AND METHODS
Protein Expression and Purification. The 1-13 C and 2-13 C glucose-labeled B1 domain of Staphylococcal protein G (GB1; UniProtKB P06654) carrying the mutations T2Q, N8D, and N37D (QDD-GB1) was expressed and purified as described elsewhere. 32 It has been established that QDD-GB1 has the same structure as wild-type GB1 and that comparisons between the two variants are valid; 32 henceforth, we refer to the triple mutant simply as GB1. 1-13 C glucose labeling results in site-selective 13 C enrichment at the δ positions of Phe and Tyr residues, while 2-13 C glucose labeling results in site-selective 13 C enrichment at their ε positions. 10,12,14 NMR Sample Preparation. To induce weak alignment of the protein in the static magnetic field, samples were prepared by using dilute liquid crystalline phases consisting of hexaethylene glycol monododecyl ether (C 12 E 6 , Sigma) and n-hexanol (Fluka), which were used without further purification. C 12 E 6 was dissolved in the protein solution (2.3 mM of 1-13 C or 3.3 mM of 2-13 C glucose labeled GB1) in H 2 O containing 10% D 2 O to reach a C 12 E 6 /water ratio of 8 wt %. 33 The pH was adjusted to 7.0, followed by addition of n-hexanol to a C 12 E 6 /n-hexanol molar ratio of 0.64. The alcohol was added in microliter steps under vigorous mixing and shaking of the solution at a temperature of 35°C. After cooling below 10°C , the solution became opalescent and stably monophasic and was stored at 4°C. Aqueous GB1 samples were dissolved to a concentration of 1 mM in 20 mM HEPES, 90% H 2 O/10% D 2 O, with small amounts of NaN 3 . The pH was adjusted to 7.0 in the sample. NMR Spectroscopy. All experiments were performed at a static magnetic field strength of 14.1 T. Experiments on the 1-13 C glucose-labeled sample were performed on a Bruker Avance IV spectrometer equipped with a cryoprobe, while RDC measurements on the 2-13 C glucose-labeled sample were performed on a Bruker Avance III spectrometer equipped with a room temperature probe. Aromatic L-optimized TROSYselected 13 C R 1ρ relaxation dispersion experiments 16 were performed with the spin-lock placed on-resonance with F52δ at temperatures of 5, 10, and 15°C. In short, the pulse sequence selects the TROSY component by using an S 3 E element, 34 and the relaxation period is divided into two blocks separated by a S 3 CT selective inversion element. 35 In each relaxation block, 13 C magnetization was aligned along the B 1 field axis by using a 4 ms tan/tanh adiabatic ramp, 36 spinlocked on-resonance using spin-lock field strengths of ω 1 /2π = {923, 1259, 1688, 2048, 2610, 3784, 4313, 5486} Hz, and finally returned to the original state by a time-reversed adiabatic ramp. Two separate experiments were acquired with and without the spin-lock relaxation period for each effective field, from which relaxation rate constants were calculated as R 1ρ = ln(I rlx /I ref )/t rlx , where I rlx and I ref are the intensities measured in the spectra acquired with and without the relaxation period, respectively, and t rlx = 20 ms is the length of the relaxation period. Relaxation dispersion data were acquired as an interleaved pseudo-3D experiment, where the effective field strengths were varied prior to incrementing the indirect evolution period t 1 .
Relaxation Data Analysis. NMR spectra were processed with NMRPipe 37 and analyzed with PINT. 38 The onresonance R 1ρ relaxation rate constant is the sum of the intrinsic transverse relaxation rate constant and the exchange contribution: R 1ρ = R 2,0 + R ex . Miloushev and Palmer have derived a general expression for symmetric two-state exchange 39 that is applicable to the case of aromatic ring flips, which involves fixed populations, p a = p b = 0.5. In the present case, where the spin-lock is applied on-resonance with the average signal, the exchange contribution can be expressed as where Δω is the difference in resonance frequency between the two sites, ω a and ω b are the effective field strengths at the resonance frequency of each site (i.e., ω a 2 = ((ω a,RDC /2 + Ω a ) 2 + ω 1 2 ), and vice versa for ω b ), Ω a is the chemical shift (Larmor frequency) offset from the carrier, ω a,RDC is the RDC, ω 1 is the spin-lock field strength (all expressed in units of rad/s), and k = 2k flip , with k flip being the ring flip rate. Data sets were fitted simultaneously, while imposing the restrictions k flip (T high ) > k flip (T low ) and R 2,0 (T high ) ≤ R 2,0 (T low ). 20 In addition, the restriction Δω(T high ) < Δω(T low ) was used, which takes into account the reduced alignment strength at higher temperatures. 33 T high and T low denote the sample temperatures of the relaxation dispersion experiments. Errors in the fitted parameters were estimated by using Monte Carlo simulations. 40 RDC Measurements and Structure-Based Predictions. Backbone 13 C α − 1 H α RDCs and aromatic 13 C− 1 H RDCs were measured in non-decoupled, non-constant time 1 H− 13 C HSQC spectra, with and without alignment medium, at 5°C . RDCs were measured for 11 clearly resolved cross-peaks corresponding to sites T17α, T18α, A26α, K31α, W43-(α,η2,ε3,ζ2,ζ3), T51α, and F52α. The resulting RDCs were used to calculate the alignment tensors (AT) based on the 1.92 and 1.14 Å X-ray crystal structures (PDB entities 1PGB 41 and 2GI9, 42 respectively), with the program REDCAT, 43 provided in NMRbox. 44 In REDCAT five independent RDCs in combination with the proteins structure are sufficient for AT calculation. 43 Its robustness was checked by systematically excluding each of the 11 RDCs, leading to the same results within margin of error. Aromatic RDCs in Tyr and Phe residues were predicted based on the experimentally determined AT and the crystal structures. Errors in the predicted RDCs were estimated as one standard deviation of RDCs predicted from 200 replicate REDCAT calculations.
The difference between the 1 H− 13 C RDC values predicted for the individual symmetry-related sites (δ1 and δ2) can be compared with the value (Δω RDC ) determined experimentally from the RDC-mediated relaxation dispersion data /2 The factor 1/2 arises because only one-half of the RDC affects the TROSY line, while the other half affects the anti-TROSY line. The absolute value arises from Δω 2 (see eq 1). The averaged value of the RDC measured in the spectrum is given by RDCs for δ1 and δ2 were calculated as a function of the χ 2 dihedral angle, as follows. Using both the 1PGB and 2GI9 structures, the atomic coordinates of the F52 aromatic ring were updated following stepwise rotation by 2°around the C β −C γ −C ζ axis, while keeping all other atom coordinates constant. The PDB coordinates were saved after each step to cover in total a rotation of 360°. On the basis of these hypothetical, intermediate state structures and the previously calculated AT, 13 C− 1 H RDCs were calculated by using REDCAT for each of the 180 different χ 2 dihedral angles.

■ RESULTS AND DISCUSSION
In isotropic solutions, aromatic ring flips of Phe and Tyr residues can normally be studied only if they possess different chemical shifts in the two δ or ε positions; although in special cases it can be possible to measure ring flips rates even if the shifts are identical. 22 In total, there are four sites to study: 13 Cδ and 13 Cε ( Figure 1A) and 1 Hδ and 1 Hε ( Figure 1B). Because of the narrow chemical shift range of aromatic nuclei and the strong coupling between them, relaxation dispersion experiments require site-selective 13 C labeling to probe the carbons 7,10−14 and additional 2 H labeling of vicinal sites to probe the protons. 15,18,45 It is not uncommon that only a subset of the four sites can be studied, either because the chemical shifts of the two symmetric positions of the ring are identical or because a site is severely broadened by exchange under the desired experimental conditions. In GB1 the chemical shifts have been measured for three aromatic residues under slow exchange conditions induced by low temperature and high pressure. 20 Y3 displays chemical shift differences for all four sites: 1 Hδ, 13 Cδ, 1 Hε, and 13 Cε. F30 displays chemical shift differences for 1 Hδ, 13 Cδ, and 1 Hε, with the δ position being severely broadened throughout the permissible temperature range from 1°C to the onset of unfolding at 40°C. F52 only displays chemical shift differences for 13 Cε, but not for the other nuclei.
Here we take advantage of the fact that the RDC usually differs between the two sides of the ring ( Figure 1C). By studying RDC-mediated exchange line broadening using relaxation dispersion methods, we increase the number of possible probes for characterizing ring flips from four to six (i.e., four chemical shifts plus two RDCs). Moreover, RDCs allow for a direct structural interpretation, which is rarely possible based on chemical shifts, except in cases where the chemical shift difference between the symmetry-related sites has been measured under slow exchange conditions. We used F52 13 Cδ as a critical test case because the δ1 and δ2 carbons have identical chemical shifts (Δω iso = 0) and therefore yield flat 13 C relaxation dispersion profiles in isotropic solutions (Figure 2A). 13 C R 1ρ Relaxation Dispersion. Aromatic 13 C R 1ρ relaxation experiments performed on F52δ in C 12 E 6 /n-hexanol liquid crystalline medium 33 reveals a significant dispersion profile, even though the 13 C chemical shift difference is zero, 20 as also evidenced by a flat dispersion profile in isotropic solvent ( Figure 2A). In contrast, Y33δ, which undergoes fast ring flips, displays flat dispersion profiles independent from the alignment medium. This result directly points to a significant difference in RDC between the two symmetry-related positions of the aromatic ring that enables a reliable fit of exchange rates ( Figure 2B).

RDC-Mediated Exchange Line Broadening Studied by
Fitting eq 1 to the experimental data yields exchange rates that agree well with previously determined values obtained from 13 C R 1ρ data for the F52ε position (Table 1), thereby validating RDC-mediated exchange as a method to study   The Journal of Physical Chemistry B pubs.acs.org/JPCB Article aromatic ring flips. The relaxation dispersion profiles are more pronounced at lower temperatures ( Figure 2B), in agreement with the expectation that decreased rates of ring flipping lead to greater exchange contributions to R 1ρ (cf. eq 1). In addition, the alignment is weaker at higher temperature, leading to reduced R 1ρ as a consequence of reduced Δω RDC , as can also be seen from the lower average RDC value, ⟨ω RDC ⟩, measured in the 1 H− 13 C HSQC spectrum (Table 1). Unfortunately, none of the other aromatic residues in GB1 are amenable to RDC-mediated exchange characterization for the following reasons: Y3 has the lowest Δω RDC value and displays significant differences in isotropic 13 C chemical shifts (Δω iso = 210 and 316 Hz at 14.1 T), which renders the contribution from the RDC-mediated broadening relatively small; Y33 is surface exposed and exhibits very fast ring flips; and F30 and Y45 are severely broadened at conditions optimal for R 1ρ experiment. The latter three cases represent general challenges in studying ring flips that are independent of the RDC approach. In fact, our approach should be widely applicable to aromatic residues suitable for characterization by relaxation dispersion experiments because Δω RDC is expected to be sufficiently large in the majority of cases. As an example, we calculated Δω RDC values for all δ and ε positions of the Tyr and Phe residues in GB1, based on the crystal structure and the determined alignment tensor. The predicted Δω RDC values range between 46 and 97 Hz, which are large enough to produce significant relaxation dispersion profiles provided that the exchange rate falls in a suitable regime. For comparison, these Δω RDC values correspond to 13 C chemical shift differences of 0.3 and 0.65 ppm at a magnetic field strength of 14.1 T. In rare cases the 1 H− 13 C bond vectors of both symmetry-related sites might be aligned identically with respect to the laboratory frame, which would yield Δω RDC near zero. This problem can be circumvented by choosing an alignment medium that generates a different alignment tensor. Otherwise, we expect that RDC-mediated exchange line broadening will serve as a valuable probe of aromatic side chain dynamics.
To validate the fitted Δω RDC values, we plotted the temperature-dependent data against ⟨ω RDC ⟩, which indeed shows the expected linear correlation (Figure 3) (cf. eqs 2 and 3). By taking the linear combinations of Δω RDC and ⟨ω RDC ⟩, we extract ω δ1,RDC = 102 ± 7 Hz and ω δ2,RDC = −78 ± 7 Hz. These RDCs can be compared with values predicted from crystal structures, which provides insights into the mechanism of ring rotations, as described below.

RDC Values Determined by Relaxation Dispersion Agree with Predictions from Crystal Structures.
We interpreted the fitted Δω RDC values and ⟨ω RDC ⟩ determined at 5°C in structural terms. We first determined the alignment tensor of GB1 from the experimental RDC values and two high-resolution crystal structures, 1PGB 41 (1.92 Å) and 2GI9 42 (1.14 Å), using REDCAT. 43 On the basis of the structural coordinates and the resulting alignment tensors, we then predicted the 1 H− 13 C RDC values for the individual symmetry-related sites, ω δ1,RDC and ω δ2,RDC , of F52δ1 and F52δ2. The experimental and predicted RDCs are compared in Table 2. Excellent agreement is observed between the predicted and experimental values of Δω RDC and ⟨ω RDC ⟩ when comparing with the 1PGB crystal structure. The 2GI9 structure shows slightly less good agreement, particularly forω δ2,RDC , while the agreement for Δω RDC is still reasonable.
Importantly, the agreement between the experimental results for F52δ and the RDCs predicted from the orientations of the H−C vectors in the crystal structure clearly indicates that the observed exchange is caused by aromatic ring flips, rather than other types of conformational exchange, such as exchange between alternative ring orientations induced by changes in the χ 1 dihedral angle. While large-scale χ 1 fluctuations are not expected to occur readily for aromatic side chains that are packed tightly in the protein core, such as F52 in GB1, this type of motion is possible for solvent exposed residues. Thus, quantitative measurement of RDC-mediated exchange makes it possible to conclusively identify ring flip dynamics even for aromatic residues that undergo fast exchange on the chemical shift time scale under all attainable experimental conditions.
It should be noted that the present evidence for jumplike ring flips does not suggest that the ring does not undergo ground-state fluctuations within each rotamer well. However, the correlation times of intrawell fluctuations are expected to be much shorter (faster) than the correlation times of ring flips, which are rare events in the case of buried rings in the interior of proteins. There is a difference of approximately 10 5 between these correlation times (sub-ns and 0.1 ms in the present case). Thus, the intrawell fluctuations are completely averaged prior to each ring flip, and the ring-flip rate extracted from the relaxation dispersion data is governed by the difference between the population-weighted ground-state and transition-state free energies.
RDC-Mediated Exchange Line-Broadening Provides Evidence for Distinct Jumplike Ring Flips. It has been proposed that aromatic rings in ubiquitin undergo complete ring rotation by continuous diffusion rather than jumplike flips. 24 The distinction between jumplike flips and such "continuous diffusion" has not previously been investigated  We predicted the variation in RDC values as a function of the χ 2 dihedral angle ( Figure 4). To estimate Δω RDC and ⟨ω RDC ⟩ values resulting from continuous diffusive motion around χ 2 , we averaged each of the two ω δ,RDC values over a complete 360°rotation by using either a uniform probability distribution (which is rather unphysical) or a probability distribution weighted by the χ 2 dihedral potential of the CHARMM36 force field with the bottom of the potential well set to either the nominal value of χ 2 = 90°or the actual value measured in the crystal structure, χ 2 = 68°(1PGB) or χ 2 = 78°( 2GI9), before calculating Δω RDC and ⟨ω RDC ⟩.
Neither the uniform nor the weighted distribution is expected to accurately portray reality because the probability distribution of ring orientations will surely depend also on tertiary interactions in the protein core. However, the greater the distinguishing features of the probability distribution, the less likely is continuous diffusional rotation. Thus, the two different averaging procedures employed here provide a first reasonable comparison of our experimental results with those expected from the continuous diffusion model. The resulting values of Δω RDC and ⟨ω RDC ⟩ are listed in Table 3. As might be expected, the continuous diffusion model yields significantly reduced Δω RDC values near zero, 46 quite different from the experimental result of Δω RDC = 90 Hz. By contrast, the Δω RDC value calculated from the ground-state structure shows excellent agreement with the value determined by experiment. In addition, ⟨ω RDC ⟩ is underestimated by the continuous diffusion model. Taken together, these results provide strong evidence that the aromatic ring of F52 in GB1 undergoes a distinct, jumplike 180°flip around the χ 2 dihedral angle.
Solid-state NMR spectroscopy can provide powerful information about aromatic ring dynamics because the dipolar coupling strength and asymmetry parameter depend on the underlying motional modes. 46−49 We note that a previous solid-state NMR study of microcrystalline wild-type GB1 indicated that the F52 ring is nearly rigid in this environment, 48 which is clearly not the case in solution as demonstrated herein. Thus, the breathing motions that enable the F52 ring to flip are apparently quenched in the crystalline state. These differences highlight the critical role of the protein packing density, where not only the local environment around the ring is important but also more remote interactions such as the protein−protein interface of the crystal.
Structural Aspects of a Ring Flips. We have previously determined the activation parameters of the F52 ring flip, including ΔH ‡ and ΔS ‡ , as well as the activation volume, ΔV ‡ , and the isothermal volume compressibility. 19 These parameters present a consistent picture of the transition state as being significantly expanded with fewer stabilizing interactions than the ground state, while populating a larger number of conformations and having a compressibility similar to that of unfolded proteins, indicating that the F52 ring flip is made possible by relatively large-scale breathing motions that resemble local unfolding. 19 Yet, our present results are fully consistent with a jumplike two-state exchange process that does not involve any intermediate or off-pathway high-energy conformations. This result contrasts with recent observations involving a buried tyrosine residue in an SH3 domain, 50 which exchanges between the ground state and an alternative highenergy conformation characterized by different values of the χ 2 dihedral angle. MD simulations showed that local breathing motions create a significant void volume (ΔV ‡ = 65 Å 3 ) allowing for rapid ring flips (k flip > 25000 s −1 ). However, once in a while (70 s −1 ) the ring flip is apparently intercepted by interactions with surrounding residues, leading to recompaction of the local structure and trapping of the ring in the highenergy conformation, which consequently is off-pathway with respect to the ring flip reaction. X-ray crystal structures of variant SH3 domains that stabilize the high-energy state provide unique structural details of large-scale structural changes that might be coupled to ring flipping. 50 The difference in behavior observed for the aromatic residues in the SH3 domain, 50 ubiquitin, 24 and GB1 illustrates the complexity and variation among proteins in the intramolecular dynamics associated with ring flipping, which evidently is critically dependent on local structure and interactions.

■ CONCLUSIONS
We have shown that RDC-mediated exchange line broadening enables measurement of aromatic ring flip dynamics using relaxation dispersion experiments. As reported previously for other nuclei, 29,30 the approach offers a valuable complement to experiments probing exchange mediated by the isotropic chemical shift. In the special case of aromatic side chains, the experimentally determined RDCs provide particularly powerful information about the mechanism of the ring flips by making it possible to distinguish between distinct jumps and alternative models, including continuous diffusive motion around the χ 2 dihedral angle as proposed by Wand and co-workers based on  studies of aromatic residues in ubiquitin. 24 We conclude that the aromatic side chain of F52 in GB1 undergoes distinct, jumplike ring flips, in full agreement with our previous conclusions based on temperature-and pressure-dependent studies of the activation volume and transition state compressibility of the exchange process. 19 ■ AUTHOR INFORMATION