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Heteronuclear proton assisted recoupling
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10.1063/1.3541251
/content/aip/journal/jcp/134/9/10.1063/1.3541251
http://aip.metastore.ingenta.com/content/aip/journal/jcp/134/9/10.1063/1.3541251
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Figures

Image of FIG. 1.
FIG. 1.

(left) Third spin assisted recoupling (TSAR) is a second-order mechanism where polarization is transferred from spin B to C using the cross term between the couplings to an assisting spin A. In the context of biological NMR, this mechanism can be used to design methods that transfer polarization between homonuclear and heteronuclear spins, referred as the PAR and PAIN-CP pulse sequence respectively. (right) PAIN-CP pulse sequence for obtaining 2D 15N–13C heteronuclear correlation spectra. The PAIN-CP mixing consists of continuous wave (CW) irradiations on 15N, 13C, and 1H channels that reintroduce second-order cross term between 15N–1H and 1H–13C dipolar couplings (terms 2 and 3) in order to transfer polarization from 15N to 13C. Note that the TSAR mechanism can be utilized both in MAS and static NMR spectroscopy.

Image of FIG. 2.
FIG. 2.

(a)–(b) Visualization of the PAIN-CP spin dynamics subspace. The space can be seen as a coupled basis between a ZQ/DQ fictitious spin I (involving the nitrogen spin N and the carbon spin C) and a proton spin H (assisting spin). The red arrows indicate the transverse PAIN-CP recoupling axis and the longitudinal off resonance contribution (from autocross terms, see Sec. II C) which result in a tilting of the effective recoupling axis. [see SI Sec. I for fictitious ZQ/DQ spin operator notations] (Ref. 26).

Image of FIG. 3.
FIG. 3.

Heteronuclear polarization transfer for the PAIN-CP conditions highlighted in the text. Simulations with all the dipolar couplings included (blue circle), and 1H–X (black dashed line) or 15N–13C (red solid line) couplings removed are considered. The spin system (top left) consists of three spin and the magnetization starts on the nitrogen and is detected on the carbon. The simulations include typical anisotropic chemical shift interactions (see Sec. II C). The rf power levels of the three CW irradiations are chosen based on optimization maps (indicated by stars on Fig. SI 2–5) (Ref. 26) and correspond to settings resulting in adequate polarization transfer efficiency. The rf power level settings (in units of the MAS frequency) are shown directly on the figure. The panels with the grey and with the white background indicate respectively conditions without and with concurrent 15N–13C CP active during the experiment.

Image of FIG. 4.
FIG. 4.

15N–13C PAIN-CP polarization transfer after 3 ms irradiation for δp0, i.e., p N = p C = p, as a function of the proton and carbon/nitrogen rf field strengths in unit of the MAS spinning frequency. The spin system is composed of three spins: a nitrogen N, an amide proton Hn, and a carbon Cα. The chosen geometry and chemical shifts are those of a typical NHnCα system found in a protein [see Sec. III C for details]. 15N, 13C, and 1H spins are irradiated on resonance. 15N–13C analytical polarization transfer maps with (a) only the TSAR term [see Eqs. (11)–(13)], (b) with the TSAR term and the TSAR autocross terms [see Eqs. (23) and (24)], (c) the TSAR term, the TSAR autocross terms and CS autocross terms [see Eqs. (23) and (24)]. 15N–13C numerical polarization transfer maps with (d) all interactions included, (e) only dipolar couplings included, (f) all interactions included except the 15N–13C coupling.

Image of FIG. 5.
FIG. 5.

15N–13C PAIN-CP polarization transfer after 3 ms irradiation for δp0, i.e., p N = p C = p, as a function of the proton and carbon/nitrogen rf field strengths in unit of the spinning frequency. The spin system is composed of three spins, a nitrogen N, an amide proton Hn, and a carbonyl carbon C. The chosen geometry and the chemical shifts are those of a typical C′NH n system found in protein (see Sec. III C for details). 15N, 13C, and 1H spins are irradiated on resonance. 15N–13C analytical polarization transfer maps with (a) only the TSAR term [see Eqs. (11)–(13)], (b) with the TSAR term and the TSAR autocross terms [see Eqs. (23) and (24)], (c) the TSAR term, the TSAR autocross terms and chemical shift autocross terms [see Eqs. (23) and (24)]. 15N–13C numerical polarization transfer maps with (d) all interactions included, (e) only dipolar couplings included, (f) all interactions included except the 15N–13C coupling.

Image of FIG. 6.
FIG. 6.

15N–13C δp0 PAIN-CP polarization transfer at ω H0 /2π = 750 MHz and ω r /2π = 20 kHz for three different 13C offset frequencies, 40 ppm (a) and (b); 177 ppm (c) and (d); 110 ppm for (e) and (f). The left column correspond to favorable low power PAIN-CP settings whereas the right column to higher power PAIN-CP settings. The spin system is extracted from the Crh x-ray structure (PDB ID: 1mu4) (Ref. 30) and is composed of L63N, four carbons (L63Cα, L63Cβ, L63Cγ, T62C), and the four protons mainly involved in the spin dynamics (L63H, L63Hα, L63Hβ2, L63Hγ). The 13C chemical shifts are taken from the protein assignment (Ref. 31). The L63N and the protons are irradiated on resonance. The simulation includes typical CSA tensor parameter for nitrogen (99 ppm, 0.19), carbonyls (−76 ppm, 0.9) and proton (5 ppm, 0.7).

Image of FIG. 7.
FIG. 7.

Figure illustrating the flexibility of PAIN-CP pulse sequence. Using an appropriate combination of rf strength, offset and mixing time PAIN-CP can accomplish: (a) selective N–C′ recoupling, (b) selective N–Cα recoupling, (c) broadband 15N–13C recoupling with contacts ranging from one-bond to long range, (d) band-selective 15N–13C recoupling to aliphatic carbons (with contacts ranging from one-bond to long range), (e) band-selective 15N–13C recoupling to aliphatic carbons except Cα's (with contacts ranging from two bond to long range). All spectra were obtained on NAc-[U-13C,15N]-f-MLF-OH at ω 0H /2π = 750 MHz and ωr/2π = 20 kHz using eight scans per t1 point. Specific δp0 PAIN-CP settings were: (a) 8 ms mixing time with ω 1C/N /2π ∼15 kHz, ω 1H /2π ∼57 kHz with the 13C offset in the middle of C′ region, (b) 1 ms mixing time with ω 1C/N /2π ∼4 kHz, 1H 43 kHz with the 13C offset in the middle of the Cα region, (c) 3 ms mixing time with ω 1C/N /2π ∼53 kHz, ω 1H /2π ∼78 kHz with the 13C offset in the middle between C′ and Cα region, (d) 9 ms mixing time with ω 1C/N /2π ∼15 kHz, ω1H/2π ∼time with ω 1C/N /2π ∼15 kHz, ω 1H /2π ∼57 kHz with the 13C offset at 28.8 ppm.

Image of FIG. 8.
FIG. 8.

15N–13C δp 0 PAIN-CP polarization transfer for p C = p N = 0.75 and p H = 1.8 (a) and p C = p N = 2.9, p H = 1.5 (b). 15N–13C δp 0 DCP polarization transfer for p C = 3.5, p N = 2.5 and p H = 10 (c) and p C = 3.5, p N = 2.5, p H = 5 (d). Both sets of simulations were performed at ω 0H /2π = 750 MHz and ω r /2π = 20 kHz. The spin system is extracted from the Crh x-ray structure (PDB ID: 1mu4) (Ref. 30) and is composed of L63N, three carbonyls (V61C′, T62C′, and L63C′), and the four protons mainly involved in the spin dynamics (T62H, T62Hα, L63H, and L63Hα). The chemical shifts are taken from the protein assignments (Ref. 31). The 13C carrier offset is set at 177 ppm, L63N, and the protons are irradiated on resonance. The simulation includes typical CSA tensor parameter for nitrogen (99 ppm, 0.19), carbonyls (−76 ppm, 0.9) and proton (5 ppm, 0.7).

Image of FIG. 9.
FIG. 9.

(a) 15N–13C TEDOR and (b) 15N–13C δp0 PAIN-CP 2D correlation experiments of [1,3]-13C GB1. (c) Expansion of the PAIN-CP spectrum. The TEDOR experiment was performed at ω 0H /2π = 750 MHz and ω r /2π = 12.5 kHz; PAIN-CP was performed at ω 0H /2π = 900 MHz and ω r/2π = 20 kHz. The TEDOR mixing was optimized to 1.4 ms to maximize the one-bond transfer, and the PAIN-CP mixing time was set to 5 ms according to simulations reported in Fig. 8.

Image of FIG. 10.
FIG. 10.

Backbone nitrogen to sidechain carbons polarization transfer: (a) 15N–13C δp0 PAIN-CP at ωr/2π = 20 kHz with p C = p N = 2.6 and p H = 2.4. (b) NCACX with DCP (ω 1C /2π = 25 kHz, ω 1N /2π = 35 kHz and 100 kHz 1H decoupling) followed by DARR (ω 1H /2π = 10 kHz) mixing at ω r /2π = 10 kHz. (c) NCACX with DCP (ω 1C /2π = 30 kHz, ω 1N /2π = 50 kHz and 100 kHz 1H decoupling) followed by CM5RR (ω 1C /2π = 100 kHz, phase ±11.46°) mixing at ω r /2π = 20 kHz. Simulations were performed at ω 0H /2π = 750 MHz. The spin system [inset of panel (a)] is extracted from the Crh x-ray structure (PDB ID: 1mu4) (Ref. 30) and is composed of L63N, four aliphatic carbons L63Cα, L63Cβ, L63Cγ, T62Cα (grey atoms) and the four protons mainly involved in the spin dynamics (T62Hα, L63H, L63Hα, L63Hβ2, L63Hγ - white atoms). The chemical shifts are taken from the protein assignment (Ref. 31). L63N and the protons are irradiated on resonance. The 13C carrier frequency is set on resonance with L63Cα. The simulation includes typical CSA tensor parameter for nitrogen (−115 ppm, 0.2), aliphatic carbons (20–25 ppm, 0.0), and proton (5.7 ppm, 0.65).

Image of FIG. 11.
FIG. 11.

ZQ/DQ heteronuclear polarization transfers between long distant spins for various 15N–13C PAIN-CP/CP conditions. The spin system is composed of three spins (a directly bonded NH pair and a remote carbon with r NC = 4.5 Å). The initial magnetization is placed on the nitrogen and is detected on the carbon. The simulations include typical CSAs (see Sec. III). The irradiation is on resonance for all spins. The rf power levels are same as in Fig. 3: p C = p N = 2.9 p H = 2.45 for 15N–13C δp0 PAIN-CP; p C = 0.36, p N = 2.64, p H = 2.05 for 15N–13C σp3 PAIN-CP; p C = 3.5, p N = 2.5, p H = 10 for σp -1 15N–13C CP; p C = 3.5, p N = 2.5, p H = 1.9 for 15N–13C CP + δp−1 PAIN-CP.

Image of FIG. 12.
FIG. 12.

The spin system used in the simulations is composed of one 15N spin, one 1H spin (which are fixed in space), and one 13C spin which position is defined on a 3 Å radius sphere by the θ and ϕ spherical coordinates with origin at the 1H or 15N in the left/right column, respectively. The spherical map represents the 15N–13C polarization transfer efficiency as a function of the position of the 13C spin for a PAIN-CP mixing time of 10 ms using p C = p N = 2.9 and p H = 2.55. The map below represents the 15N–13C polarization efficiency for ϕ = 0 as a function of the mixing time and the θ angle.

Image of FIG. 13.
FIG. 13.

Long distance 15N–13C polarization transfers for various 15N–13C PAIN-CP/CP conditions. The spin system is composed of four nuclear spins (a nitrogen with directly attached proton, directly bonded Cα and a remote carbon with rNC = 4.5 Å). The magnetization starts on the nitrogen and is detected on the carbon. The simulations include typical CSAs (see Sec. III B). The triple irradiation is performed on resonance for each spin. The rf power levels are the same as settings used in Fig. 3: p C = p N = 2.9 p H = 2.45 for δp 0 PAIN-CP in (a); p C = 0.36, p N = 2.64, p H = 2.05 for σp 3 PAIN-CP in (b); p C = 3.5, p N = 2.5, p H = 10 for δp −1 15N–13C CP in (c), and p C = 3.5, p N = 2.5, p H = 1.9 for 15N–13C CP + δp−1PAIN-CP in (d).

Image of FIG. 14.
FIG. 14.

2D 15N–13C correlation PAIN-CP spectra on [U–13C,15N] (a) and heterogeneously 50%/50% [U–13C]/[U–15N] labeled (b) Crh. (a) was obtained at ω 0H /2π = 750 MHz, ω r /2π = 20 kHz with 15 ms mixing time. (b) was obtained at ω 0 H/2π = 900 MHz, ω r /2π = 20 kHz. The spectrum in (b) is a sum of experiments with mixing time of 5 and 10 ms. (c) Crh x-ray structure [PDB entry: 1MU4 (Ref. 30)] and (d) solid-state NMR structures of an isolated monomer. 15N–13C PAIN-CP buildup curves for the spin system [see panel (e)] composed of one nitrogen, three carbons, and five protons without (e) and with fast methyl rotation (f). The magnetization starts on the L63N spin and is distributed to the L63Cα (directly bonded), the L35Cδ2 (4.26 Å distant), the L35Cγ (5.59 Å distant). The three rf power levels of the CW irradiations are chosen based on optimization maps (see Fig. SI 2) (Ref. 26). The rf power level settings (in units of the MAS frequency) are p C = p N = 2.9 p H = 2.45 (δp 0 PAIN-CP).

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/content/aip/journal/jcp/134/9/10.1063/1.3541251
2011-03-01
2014-04-19
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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Heteronuclear proton assisted recoupling
http://aip.metastore.ingenta.com/content/aip/journal/jcp/134/9/10.1063/1.3541251
10.1063/1.3541251
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