Pre-Homonuclear Decoupling Enables High-Resolution NMR

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Pre-Homonuclear Decoupling Enables High-Resolution NMR Analysis of Intrinsically Disordered Proteins in Solution Jonghyuk Im, Jongchan Lee, and Jung Ho Lee J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.9b01773 • Publication Date (Web): 01 Aug 2019 Downloaded from pubs.acs.org on August 2, 2019

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The Journal of Physical Chemistry Letters

Pre-Homonuclear Decoupling Enables High-Resolution NMR Analysis of Intrinsically Disordered Proteins in Solution Jonghyuk Im, Jongchan Lee, and Jung Ho Lee* Department of Chemistry, Seoul National University, Seoul 08826, Korea AUTHOR INFORMATION Corresponding Author *[email protected]

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ABSTRACT Probing the atomic details of intrinsically disordered proteins is crucial to understanding their biological function and relation to pathogenesis. Although amide-detected NMR experiments are widely employed in protein studies, 3JHNH couplings between amide (1HN) and alpha (1H) protons impose an intrinsic limit on the achievable 1HN linewidth. Here, we present a homonuclear decoupling method that narrows the alpha-synuclein 1HN linewidths to 3–5 Hz. Tightly distributed 1JCH coupling values were employed to generate homogeneous anti-phase coherences of 2HzHNy and 4H(2)zH(3)zHNy for non-glycine and glycine residues, respectively, which were combined with their in-phase HNy counterparts to achieve homonuclear decoupling. By reducing the multiplet structure to a singlet, the width of the 1HN crosspeak was reduced by ~3-fold in the 2D HSQC and 3D intra-HNCA spectra, and good spectral quality was achieved without the need for post-processing.

TOC GRAPHICS

KEYWORDS NMR resolution, linewidth narrowing, homonuclear decoupling, intrinsically disordered proteins, PHD-HSQC


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Intrinsically disordered proteins (IDPs) are abundant in the eukaryotic proteasome and are closely related to the pathogenesis of many degenerative disorders1,2. Because most tools of structural analyses target well-defined structures rather than disordered conformations, nuclear magnetic resonance (NMR) provides a unique way to investigate the atomic details of IDPs in solution3,4. The NMR peaks of IDPs are sharp and strong, while their spectral dispersion is very poor. Thus, IDP signals tend to substantially overlap, and resolving them is crucial for the NMR analysis of IDPs. Amide-detected NMR experiments are widely employed in protein NMR5,6. Unlike in the indirect dimension, simply extending the chemical shift evolution time in the direct dimension leads to multiplet splitting rather than linewidth narrowing, due to the concurrent 3JHNH evolution between the amide (1HN) and alpha (1H) protons. Moreover, resonance linewidths (i.e. full width at half maximum, FWHM) in the 1H dimension are generally a few times broader than those in the

15N

dimension for

IDPs, provided that the signal decay is fully sampled in both dimensions. Thus, an efficient 3JHNH decoupling method potentially provides a dramatic improvement in NMR resolution (Figure 1).

Figure 1. NMR linewidth narrowing by pre-homonuclear decoupling (PHD). 2D 1H-15N HSQC was acquired in the (a) absence and (b) presence of PHD on a

13C/15N-enriched

alpha-synuclein (S) protein in a 850 MHz


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spectrometer at 20°C. Sensitivity-enhanced (SE) HSQC was employed for the control experiment in (a). Data acquisition time was 270 ms in the direct (1HN, 𝑡2) dimension. The cross-sections of two resonances are compared in the insets. (c) 1HN linewidths, defined as the full width at half maximum (FWHM), are displayed for each S amide cross-peak of SE-HSQC (open dots) and PHD-HSQC (closed dots). No apodization was applied.

Homonuclear decoupling (also known as pure shift NMR)7-9 of protons during the free induction decay (FID) period is challenging. The excitation of protons that resonate inside a narrow bandwidth, followed by successive inversion of protons outside the bandwidth, decouples the former protons from the latter. The Zangger-Sterk method10 assigns each narrow bandwidth to different regions of the sample tube and decouples all regions simultaneously to achieve broadband decoupling. Additionally, bilinear rotation decoupling (BIRD)11-13 suppresses the J coupling evolution of the protons attached to certain heteronuclei (e.g.,

15N)

from the protons attached to other kinds (e.g.,

13C, 12C,

and

14N).

A different

approach utilizes low flip-angle hard pulses14 or chirp pulses with pulsed field gradients (PSYCHE)15,16 to achieve homonuclear decoupling based on the anti-z-COSY17 experiment. When the bandwidths of active and passive ( ± 1 and 0 coherence order) spins are well separated, efficient decoupling with superior sensitivity can be achieved by band-selective decoupling18-20. Interestingly, effective spinlocking of active spins resulted in selective homonuclear decoupling21. However, the aforementioned methods, which apply radio frequency (RF) pulses and pulsed field gradients during the FID period, are not free from periodic sidebands, linewidth broadening, and water-induced dynamic range problems, although ongoing efforts aim to reduce the artifacts22,23. In-phase/anti-phase (IPAP)24 is another widely employed method to decouple homonuclear spins. IPAP approach is based on the acquisition of in-phase (IP) and anti-phase (AP) spectra, that are combined to provide a spectrum only with the downfield (or upfield) component of multiplets. Decoupling artifacts are minimal in IPAP experiments because no RF pulses or gradients are applied during the FID period. This approach is routinely used to decouple alpha (13C) and carbonyl (13C′) carbons in 13C′-detected IDP experiments25-27 by combining the C′y IP and 2CzC′y AP coherences. As


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1J C’C

values are narrowly distributed (50–55 Hz)28, a single representative 1/(21JC’C) period can be

chosen to create homogeneous 2CzC′y coherences, which upon uniform scaling, match the intensities of C′y for all residues. On the other hand, 3JHNH values are sensitive to protein backbone conformation and span a wide range of 2–10 Hz29-31, or 5–8 Hz in IDPs32 (Figure 2). Thus, a single 3JHNH coupling evolution period cannot be chosen to generate homogeneous AP coherences for all amides.

Figure 2. Relative dispersion of the 3JHNH and 1JCH values for the non-proline residues of ubiquitin (blue dots)28,33 and S (open red dots)32 proteins. The wide dispersion of 3JHNH coupling values prevents the selection of a single evolution period (1/23JHNH) to generate homogeneous AP.

By employing the intra-HNCA scheme34-36, we utilized the narrowly distributed 1JCH coupling values (141–144 Hz for IDP non-proline residues, Figure 2) to generate homogenous HN AP coherences with respect to the intra-residue H (i.e., we created 2HzHNy coherences; and 4H(2)zH(3)zHNy for Gly residues). These AP coherences matched and canceled all but one of the multiplet lines of the IP HNy coherences to achieve homonuclear decoupling. Because the Hz terms were introduced by the 1JCH coupling evolution, which occurs long before the FID period, we named this method the “prehomonuclear decoupling” (PHD). We first investigated the effect of PHD on the 2D 1H-15N heteronuclear single quantum coherence (HSQC) spectra (Figure 1). The

13C/15N-enriched

alpha-synuclein (S) protein was employed as our

model IDP. NMR samples contained 0.5 mM of 13C/15N-enriched S, 20 mM NaCl, and 20 mM sodium phosphate buffer (pH 6.0) in H2O/D2O (95%/5%). The control experiment is a sensitivity-enhanced (SE) HSQC37-40, with the 1H composite pulse decoupling during the 15N evolution period41. 2D HSQC spectra in the absence and presence of PHD are displayed in Figure 1a and 1b, respectively. The data ACS Paragon Plus Environment

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matrix consisted of 400 (15N, 𝑡1) × 4000 (1HN, 𝑡2) complex data points with spectral width of 1920 Hz ( 𝑡1) and 14800 Hz (𝑡2). In order to minimize the biased appearance of overlaps, contour levels were

adjusted such that the

15N

linewidths appear the same in the control and PHD spectra. The 1HN

linewidths (i.e. FWHM) strongly decreased from 9–14 Hz in the SE-HSQC to 3–5 Hz in the PHDHSQC (Figure 1c). The extent of line narrowing was directly proportional to the 3JHNH coupling values (Supporting Information, Figure S1). Resonance cross-sections in Figure 1 illustrate how triplets of glycine and doublets of non-glycine residues collapsed into sharp singlets. Pre-homonuclear decoupling was implemented using the pulse sequence shown in Figure 3. Detailed parameters are provided in Figure S2. The main strategy was to introduce the intra-residue Hz terms during the d→e period by taking advantage of the uniform 1JCH coupling values, and then carefully transfer these terms to the end of the pulse sequence to generate AP coherences for decoupling.

Figure 3. PHD-HSQC pulse sequence. Thin and thick black rectangles represent 90° and 180° hard pulses, respectively. Lobes with s, e, r, and i represent the sinc, eburp, reburp, and iburp-shaped pulses,42 where

the reburp and eburp pulses were selectively applied to amide protons. The presence and absence of the two 1H 180° pulses in the brackets generate the AP and IP magnetizations, respectively. Delays (ms): 𝜏1 = 2.7; 𝜏2 = 25; 𝜏3 = 17.5; 𝜏4 = 4.7; 𝜏5 = 1.7. Phase cycles: 𝜙1 = 𝑥, ― 𝑥; 𝜙2 = 𝑥; 𝜙3 = 𝑦+52°; 𝜙4 = 45°, 45°, 135°, 135° (AP) and 𝜙4 = 𝑥,𝑥, ― 𝑥, ― 𝑥 (IP); 𝜙5 = 𝑦+52°; 𝜙𝑟𝑒𝑐 = 𝑥, ― 𝑥, ― 𝑥,𝑥. Quadrature in (15N, 𝑡1) was achieved by States-TPPI.43

The evolution of the magnetization from point a to d in Figure 3 is summarized below. The J coupling Hamiltonian (Hz) and its evolution time are written above each arrow. The index of only the previous


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residue is shown as (𝑖-1). The trigonometric terms from the 15N chemical shift evolution and the signs of the product operators were omitted for simplicity. 1

a. 𝐻𝑧

𝐽𝐻𝑁𝐻𝑧𝑁𝑧; 2𝜏1 = 1/(21𝐽𝐻𝑁)

1

b. 2𝐻𝑧𝑁𝑧

𝐽𝐻𝑁𝐻𝑧𝑁𝑧; 2𝜏1 = 1/(21𝐽𝐻𝑁)

1

𝐽𝐶𝛼𝑁𝐶𝛼𝑧𝑁𝑧; 2𝜏2 = 1/(21𝐽𝐶𝛼𝑁)

2

𝐽𝐶𝛼𝑁𝐶𝛼(𝑖 ― 1)𝑧𝑁𝑧; 2𝜏2 = 1/(21𝐽𝐶𝛼𝑁)

1

𝐽𝐶′𝑁𝐶′(𝑖 ― 1)𝑧𝑁𝑧; 2𝜏3 = 1/(21𝐽𝐶′𝑁)

𝑐. 8𝑁𝑧𝐶𝛼𝑧𝐶𝛼(𝑖 ― 1)𝑧𝐶′(𝑖 ― 1)𝑧 1

𝐽𝐶′𝐶𝛼𝐶′(𝑖 ― 1)𝑧𝐶𝛼(𝑖 ― 1)𝑧; 2𝜏4 = 1/(21𝐽𝐶′𝐶𝛼)

d. 4𝑁𝑧𝐶𝛼𝑧𝐶′(𝑖 ― 1)𝑧 After the 1JCH coupling evolution during the 2𝜏5 = 1/(21JCH) period, 𝜙4 = 45° rescued the AP magnetizations with respect to H for both 1) glycine and 2) non-glycine residues. By omitting the two 1H

pulses inside the brackets at point d, the 3) IP magnetizations were obtained. Although removing the

entire c→f period generates IP signals at an even higher sensitivity, we found that both IP and AP signals should be subject to the same relaxation pathway to achieve homogeneous AP/IP ratios across the entire protein residues. During the e→h period, the evolution of the magnetization in a→d is reversed to obtain the following magnetizations at h: 1) glycine residues, AP: 4𝐻𝛼(2)𝑧𝐻𝛼(3)𝑧𝐻𝑦; 2) non-glycine residues, AP: 2𝐻𝛼𝑧𝐻𝑦; 3) all residues, IP: 𝐻𝑦,


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where H indicates the alpha proton attached to non-glycine C, whereas H(2) and H(3) are the two alpha protons attached to glycine C. The pulse sequence was designed to add 1) and 3) for glycines, and 2) and 3) for non-glycines, without the need for post-processing. Figure 4 shows a region of the PHD-HSQC spectrum (Figure 1b) to illustrate how IP and AP signals are combined to decouple glycine and non-glycine residues, simultaneously. Note that the 1HN chemical shift of a non-glycine residue is shifted by half of the 3JHNH value in the PHD spectrum.

Figure 4. Combining IP and AP signals for homonuclear decoupling. IP, AP, and (IP + AP) spectra were acquired on the S protein in separate experiments and displayed with small offsets in the 15N dimension. Blue and red colors indicate positive and negative peaks, as shown in the cross-sections.

Careful balancing of IP and AP coherences was crucial to obtaining resonance lineshapes without distortion. The AP coherence was decreased by

2 compared with its IP counterpart by setting 𝜙4 =

45°, and additional signal loss occurred during the e→h period due to the T1 relaxation of alpha protons41. Therefore, for each increment, two scans of IP and four scans of AP signals were combined after downscaling the AP signals by 15% by setting 𝜙2 = 32°. This whole process was embedded in the pulse sequence for convenience, resulting in less than a 10% decrease in sensitivity compared with the regular IPAP post-processing. The (IP+AP) and (IP-AP) signals could not be combined to gain


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additional sensitivity, due to the wide dispersion of 3JHNH values, but the generation of two separate spectra with comparable signal-to-noise (SNR) can aid the spectral analysis of weak signals. In the 15N dimension, the mixed-time evolution was employed for optimal sensitivity and resolution44. Evolving the 15N chemical shifts during the b→c rather than f→g period matched the 15N linewidths of IP and AP coherences, while preventing the use of the SE scheme. The sensitivity or SNR per unit time of the PHD-HSQC was 11% (mean) compared to its control SEHSQC (Figure S3). The decreased sensitivity of the PHD method can be ascribed to the lengthy transverse relaxation time, lack of the SE scheme, splitting of AP magnetizations by setting 𝜙4 = 45°, fast relaxation of the AP magnetizations during the e→h period, and increased number of RF pulses. However, enhancement of the NMR resolution at the price of sensitivity may be a reasonable trade-off for IDPs. For instance, when PHD-HSQC was acquired for 2.5 hours on a 0.5 mM S protein inside a 850 MHz spectrometer equipped with a z-gradient cryoprobe (Figure 1b), the spectral overlaps in all types of residues were greatly resolved (Figure 5), while the observed SNR of ca. 300 was good for most quantitative analyses. PHD with higher sensitivity can be achieved when exclusively considering non-glycine (𝜙4 = 90° for AP) or glycine (𝜙4 = 0°, 𝜏5 = 1/(81JCH)) residues.

Figure 5. Comparison of signal overlap in the SE-HSQC and PHD-HSQC spectra. Each panel displays an expanded view of the spectra in Figure 1.


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The utility of the PHD scheme can be further highlighted in multidimensional NMR experiments. For example, 13C′, 13C, or 1H chemical shifts can be obtained by evolving coherences during the c→d or d→e periods, or by inserting an 1H evolution period at point e, all of which have sensitivities comparable to PHD-HSQC. On the other hand, their control experiments of 3D HNCO, intra-HNCA, or intra-HNHA are considerably less sensitive than SE-HSQC. Therefore, 60% of the NMR sensitivity was retained in the 3D PHD-intra-HNCA experiment compared to its control (Figure S3), and most peaks were resolved only in the PHD spectrum (Figure 6). It is clear that the proposed decoupling method can be routinely applied to many multidimensional NMR experiments.

Figure 6. Effect of PHD on the 3D intra-HNCA spectra. 2D slices at a fixed 13C chemical shift are shown. NMR data were acquired on S using the same parameters as in Figure 1, except that the acquisition times in the 1H, 15N,

and 13C dimensions were set to 270, 21, and 13 ms, respectively, with the total data acquisition time of 23

hours for each 3D experiment.

1HN

linewidth narrowing was also observed in the small well-ordered ubiquitin protein, upon applying

the PHD scheme (Figure S4). However, few residues displayed small peak shoulders in the 1H dimension (Figure S4), likely due to the imbalanced relaxation between AP and IP. This effect was also observed in S, to a lesser extent (Figure S5). For different proteins, we recommend carefully adjusting 𝜙2. For example, 𝜙2 was adjusted to decrease the AP signal by 15% for S, whereas this adjustment was unnecessary for ubiquitin, reflecting the faster relaxation of AP over IP for ubiquitin during e→h. We conclude that PHD can be applied to IDPs and polypeptides with favorable relaxation properties, providing extremely narrow 1HN linewidths with minimal artifacts. The proposed decoupling scheme will facilitate the assignment and analysis of large and complex IDPs in solution. ACS Paragon Plus Environment

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ASSOCIATED CONTENT Supporting Information. Full description of the PHD-HSQC pulse sequence and the Bruker pulse program, the PHD-HSQC spectrum of ubiquitin, and the effect of PHD on peak intensities and lineshapes. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Notes The authors declare no competing financial interests. ACKNOWLEDGMENT We are grateful to Dr. Ad Bax (National Institutes of Health, USA) for his insightful comments. This work was supported by the Creative-pioneering researchers program through the Seoul National University (SNU), the Korea Basic Science Institute (KBSI) under the R&D program (Project No. D39700), and the National Research Foundation of Korea (2019R1C1C1009685). REFERENCES 1. Wright, P. E.; Dyson, H. J., Intrinsically disordered proteins in cellular signalling and regulation. Nat. Rev. Mol. Cell Biol. 2015, 16 (1), 18-29. 2. Uversky, V. N.; Oldfield, C. J.; Dunker, A. K., Intrinsically disordered proteins in human diseases: introducing the D2 concept. Annu. Rev. Biophys. 2008, 37, 215-246. 3. Lee, J. H.; Li, F.; Grishaev, A.; Bax, A., Quantitative residue-specific protein backbone torsion angle dynamics from concerted measurement of 3J couplings. J. Am. Chem. Soc. 2015, 137 (4), 14321435. 4. Jensen, M. R.; Ruigrok, R. W.; Blackledge, M., Describing intrinsically disordered proteins at atomic resolution by NMR. Curr. Opin. Struct. Biol. 2013, 23 (3), 426-435. 5. Grzesiek, S.; Bax, A., Correlating backbone amide and side chain resonances in larger proteins by multiple relayed triple resonance NMR. J. Am. Chem. Soc. 1992, 114 (16), 6291-6293. 6. Sattler, M.; Schleucher, J.; Griesinger, C., Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution. Prog. Nucl. Magn. Reson. Spectrosc. 1999, 34, 93-158. ACS Paragon Plus Environment

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31. Marion, D.; Wüthrich, K., Application of phase sensitive correlated spectroscopy (COSY) for measurements of proton-proton spin-spin coupling constants in proteins. Biochem. Biophys. Res. Commun. 1983, 113, 967-974. 32. Mantsyzov, A. B.; Maltsev, A. S.; Ying, J.; Shen, Y.; Hummer, G.; Bax, A., A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein, an intrinsically disordered protein. Protein Sci. 2014, 23 (9), 1275-1290. 33. Maltsev, A. S.; Grishaev, A.; Roche, J.; Zasloff, M.; Bax, A., Improved cross validation of a static ubiquitin structure derived from high precision residual dipolar couplings measured in a drugbased liquid crystalline phase. J. Am. Chem. Soc. 2014, 136 (10), 3752-3755. 34. Brutscher, B., Intraresidue HNCA and COHNCA experiments for protein backbone resonance assignment. J. Magn. Reson. 2002, 156 (1), 155-159. 35. Nietlispach, D.; Ito, Y.; Laue, E. D., A novel approach for the sequential backbone assignment of larger proteins: selective intra-HNCA and DQ-HNCA. J. Am. Chem. Soc. 2002, 124 (37), 1119911207. 36. Permi, P., Intraresidual HNCA: An experiment for correlating only intraresidual backbone resonances. J. Biomol. NMR 2002, 23 (3), 201-209. 37. Kay, L.; Keifer, P.; Saarinen, T., Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J. Am. Chem. Soc. 1992, 114 (26), 1066310665. 38. Schleucher, J.; Sattler, M.; Griesinger, C., Coherence selection by gradients without signal attenuation: application to the three-dimensional HNCO experiment. Angew. Chem. 1993, 32 (10), 1489-1491. 39. Palmer III, A. G.; Cavanagh, J.; Wright, P. E.; Rance, M., Sensitivity improvement in protondetected two-dimensional heteronuclear correlation NMR spectroscopy. J. Magn. Reson. 1991, 93 (1), 151-170. 40. Bax, A., Two-dimensional nuclear magnetic resonance in liquids. Delft Univ. Press: 1982. 41. Bax, A.; Ikura, M.; Kay, L. E.; Torchia, D. A.; Tschudin, R., Comparison of different modes of two-dimensional reverse-correlation NMR for the study of proteins. J. Magn. Reson. 1990, 86 (2), 304318. 42. Geen, H.; Freeman, R., Band-selective radiofrequency pulses. J. Magn. Reson. 1991, 93 (1), 93141. 43. Marion, D.; Ikura, M.; Tschudin, R.; Bax, A., Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins. J. Magn. Reson. 1989, 85, 393-399. 44. Ying, J.; Chill, J. H.; Louis, J. M.; Bax, A., Mixed-time parallel evolution in multiple quantum NMR experiments: Sensitivity and resolution enhancement in heteronuclear NMR. J. Biomol. NMR 2007, 37 (3), 195-204.


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Pre-Homonuclear Decoupling Enables High-Resolution NMR

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