NMR experiments

We developed a set of eight experiments, each providing a different CCR rate. In each case, only one CCR rate at a time is measured; spin evolution due to all other substantial CCR mechanisms is either refocused or leads to coherence that is not converted to observable magnetization. All experiments are four-dimensional. The directly detected nucleus is amide proton and the indirectly detected ones are amide nitrogen, carbonyl carbon, and alpha carbon. Carbon nuclei belong to the same amino acid residue as the amide group, or the preceding one, depending on the experiment. Information on measured nuclei, CCR rates, and the angles that we can extract from each rate is summarized in Table 1 and Fig. 1.

Table 1 Information on peak types, CCR rates, and anglesFig. 1

Scheme showing the nuclei and dihedral angles involved in the CCR effect for each experiment. For each CCR rate the map of the geometrical contribution (dependence on the dihedral angles \(\phi\)—shown on a horizontal axis, and \(\psi\)—shown on a vertical axis) is shown. Indices i and \(i-1\) indicate the relative positions of residues involved, in each case the signal detection was performed on the \(i^\) amide proton

In all the experiments we conducted, we used the quantitative gamma approach (Tjandra et al. 1996; Brutscher 2000), which required two versions of the experiment to be carried out: a reference version and a transfer version. We designed each pulse sequence in the same way. After the relaxation delay d1, the equilibrium \(^\)C and \(^\)N polarization was eliminated, using for each of these channels a pair of 90 degree pulses, each followed by a gradient. Then, four main steps were taken, namely:

1.

Create the coherence needed for CCR evolution

2.

CCR block: carry out the CCR evolution. At the end of it the coherence consists of two parts: ‘auto’ (identical to the initial state at the beginning of the CCR block) and ‘cross’ (originating from the CCR evolution of the original state). In the reference and transfer versions the phases of the last pulses of the block are adjusted to select the desired component: ‘auto’ and ‘cross’, respectively.

3.

Unifying block (present only if the forms of ‘auto’ and ‘cross’ coherences after the CCR block are different): convert the coherence to the same state (only differing in terms of intensity) for both versions of the experiment, reference and transfer. In the reference version this block should keep the coherence unchanged, while in the transfer version it should refocus anti-phase magnetization with respect to certain nuclei. Both versions should be as similar as possible regarding the delays and the number of pulses, to provide quantitative results of the CCR rates.

4.

Transfer the coherence to the observable magnetization and acquisition. A sensitivity enhancement block with gradient coherence selection (Kay et al. 1992; Schleucher et al. 1993) was used in each experiment, therefore the \(\textrm^\textrm_\)operator, used in the following subsections in the schemes of coherence transfer pathway description, stands for phase-modulated signal of the following form: \(^\textrm_}\sin (\varOmega _\textrm \,\textrm_3)\pm ^\textrm_}\cos (\varOmega _\textrm \,\textrm_3)}\) (where \(\varOmega _N\) is the nitrogen frequency and \(t_3\) is the nitrogen evolution time).

More detailed descriptions of all experiments are given in the subsections below.

Experiment 1. \(\textrm^_\textrm^_\) DD–\(\textrm_\textrm^}_\) DD

The first experiment measures the CCR effect between \(\textrm^_^_}\) DD and \(\textrm_)}\textrm^}_)}\) DD. It yields information on the \(\) angle (peak \(^}_)}-\textrm_)}}\) – \(\textrm'_\) – \(\textrm^\alpha _\)informs on \(}\)). Our pulse sequence is shown in Fig. 2 and is based on the standard 4D HNCOCA pulse sequence (Bax and Ikura 1991; Yang and Kay 1999). The coherence transfer before the CCR block is as follows:

$$\begin & \textrm^\textrm_\xrightarrow []} 2\textrm^\textrm_\textrm_\mathrm}\xrightarrow [}]} 2\textrm'_\textrm_\mathrm}\xrightarrow []}} \\ & \xrightarrow []}} 4\textrm^_\textrm'_\textrm_\mathrm}\Rightarrow \text \end$$

Fig. 2

Pulse sequence of the experiment 1 (\(\textrm^_\textrm^_\) DD–\(\textrm_\textrm^}_\) DD CCR). The evolution of \(^}\) is in constant-time mode during CCR evolution: \(A = (\textrm_+t_)/4\) and \(B = (\textrm_-t_)/4\), C\('\) evolution is in (semi)constant-time mode (Grzesiek and Bax 1993): \(a_2=(\varDelta _}+t_2)/2, \ b_2=t_2(1-\varDelta _})/(2t^_), \ c_2=\varDelta _}(1-t_2)/(2t^_)\) and N evolution is in (semi)constant-time mode: \(a_3=(\varDelta _}+t_3)/2, \ b_3=t_3(1-\varDelta _})/(2t^_), \ c_3=\varDelta _}(1-t_3)/(2t^_)\). The semi-constant or constant time mode is chosen automatically, based on the number of sampling points and spectral width in a given dimension. The delays were set as follows: \(\varDelta _}\)=5.4 ms, \(\varDelta _}\)=31 ms, \(\varDelta _}\)=29 ms, \(\varDelta _}\)=9.1 ms, and \(\varDelta _}\)=3.4 ms. Total CCR rate evolution time is set as \(\textrm_\)=28.6 ms. Unless noted explicitly, pulse phases are set to x. Phase \(\phi _\) depends on the version of the experiment: for reference experiment x, for transfer experiment y. The value of individual phase cycles are \(\phi _=x,-x\), \(\phi _=2(x),2(-x)\), \(\phi _=x\), \(\phi _=4(x),4(y),4(-x),4(-y)\), \(\psi =y\) and receiver \(\phi _=\phi _+\phi _+2\cdot \phi _\). Quadrature detection is achieved by States-TPPI (Marion et al. 1989) for \(^}\) (change of \(\phi _\) phase) and C\('\) (change of \(\phi _\) phase) evolutions, and echo-antiecho (Kay et al. 1992; Schleucher et al. 1993) for N evolution (change of \(\phi _\) and \(\psi\) phases and change of \(G_\) amplitude sign). Selective pulses for C\('\) and \(^}\) spins were Q3 (for 180\(^\circ\) pulses) and Q5 (for 90\(^\circ\) pulses) (Emsley and Bodenhausen 1992). The selective 180\(^\circ\) pulse on proton channel (marked \(_}\)) affects only \(^}\) and was prepared by Bruker WaveMaker tool, using offset=4.3 ppm, bandwidth=3 ppm, reburp shape (Geen and Freeman 1991). Simultaneous inversion of \(^}\) and C\('\) spins was achieved using 6-element composite pulse (Shaka 1985). The pulses labelled by a star ("*") were executed only in the transfer version of the experiment. Rectangles represent hard pulses while rounded cones symbolize shaped pulses; the empty ones are 180\(^\circ\) pulses and the filled ones are 90\(^\circ\) pulses. Gradients with numbers from 1 to 12 are cleaning gradients. Gradients \(G_\) and \(G_\) are used for coherence selection in echo-antiecho quadrature detection. Proton decoupling is performed with composite pulse scheme waltz65 (Zhou et al. 2007) and nitrogen decoupling during acquisition is performed with composite pulse scheme garp (Shaka et al. 1985). The CCR and unifying blocks are indicated by rectangle boxes

The CCR block contains two types of evolution: CCR evolution, and constant-time chemical shift evolution of \(\textrm^\alpha _\). Two substantial unwanted CCR evolutions, i.e. \(\textrm_\textrm^\textrm_\) DD–\(\textrm^_\) CSA and \(\textrm^_\) CSA–\(\textrm_\) CSA were refocused during the CCR block by the first and third 180\(^\circ\) pulses acting on nitrogen nuclei. Another unwanted CCR evolution, i.e. \(\textrm^_\textrm^_\) DD–\(\textrm_\) CSA, did occur during the CCR block, but the resulting coherences (on the schemes of coherence transfer below shown in squared brackets) were not transferred into an observable magnetization. To minimize the loss of coherence due to \(^-\textrm^}\) J-coupling evolution, we set the overall evolution time to the inverse of the \(^-\textrm^}\) J-coupling constant, that is, 28.6 ms. During the CCR block, the initial 4\(\textrm^_\) \(\textrm'_\) \(\textrm_\) coherence is partially converted into 16\(\textrm^_\) \(\textrm^_\) \(\textrm'_\) \(\textrm_\) \(\textrm^\textrm_\) coherence, so we can use different phases of the last 90\(^\circ\) pulses (acting on \(\textrm^\) and N) to select each component: The x-pulses allow us to observe the unchanged coherence, while the y-pulses allow us to observe the component originating from CCR evolution. In the undesired coherence the transverse component remains, thus it is dephased by the following gradient (G5). During the unifying block, the 16\(\textrm^_\) \(\textrm^_\) \(\textrm'_\) \(\textrm_\mathrm}\) \(\textrm^\textrm_\) five-spin order is transferred back to a 4\(\textrm^_\) \(\textrm'_\) \(\textrm_\mathrm}\) three-spin order. This is achieved by the evolution of the J\(_}\) coupling and the consecutive evolution of the J\(_^\textrm^}\) coupling; see Fig. 3.

Fig. 3

Scheme showing the difference in coherence transfer between transfer and reference version of the Experiment 1( \(\textrm^_\textrm^_\) DD–\(\textrm_\textrm^}_\) DD)

Next, during the transfer of the coherence to the observable magnetization, we allow for the evolution of the chemical shifts of \(\textrm'_\) and \(\textrm_\). The coherence transfer after the CCR and unifying blocks is as follows:

$$\begin &4\textrm^_\textrm'_\textrm_\mathrm}+ [8\textrm^_\textrm'_\textrm^\textrm_\textrm_\mathrm}] \xrightarrow []}} \\&\quad \xrightarrow []}} 2\textrm'_\textrm_\mathrm}+ [4\textrm'_\textrm^\textrm_\textrm_\mathrm}]\xrightarrow [}]}\\&\quad \xrightarrow [}]} 2\textrm^\textrm_\textrm_\mathrm}+ [\textrm_\mathrm}] \xrightarrow []} \\&\quad \xrightarrow []} \textrm^\textrm_+ [\mathrm] \end$$

Previously, this rate was measured by Reif et al. (1997) and by Yang and Kay (1998) as a 3D HN(CO)CA J-resolved experiment, and by Pelupessy et al. (1999a) in a 2D quantitative gamma version (without carbon evolution). The latter solution is similar to ours (a quantitative gamma approach with the same operator at the start of the CCR block), but there are some important differences. In the 2D version, the lack of \(\textrm^\) evolution made it possible to retain the initial coherence at the end of the CCR block in the transfer version too, as the proper shifting of the \(\textrm^\) pulses led to \(\textrm^\)- \(\textrm^\) J-coupling evolution. In a 3D version, this would require real-time \(\textrm^\) evolution (after the CCR block), which would, in our opinion, not be optimal due to the occurrence of J\(_^\textrm^}\) modulation. We therefore included C\(^\) chemical shift evolution in the CCR block and added a unifying block directly after it.

Experiment 2. \(\textrm^_\textrm^_\) DD–\(\textrm_\textrm^}_\) DD

The second experiment measures the CCR effect between \(\textrm^_\textrm^_\) DD and \(\textrm_\textrm^}_\) DD. From the peak \(^}_)}-\textrm_)}}\) – \(\textrm'_\) – \(\textrm^\alpha _)}\)we get information on the \()}}\) angle. The first measurement of this rate was proposed by Pelupessy et al. (1999b), whose 3D HNCA J-resolved experiment made it possible to simultaneously measure \(\textrm^_\textrm^_\) DD–\(\textrm_\textrm^}_\) DD and \(\textrm^_\textrm^_\) DD–\(\textrm_\textrm^}_\) DD rates. In our experiment, the coherence transfer pathway is based on a 4D HNCO\(_\)CA\(_\) experiment (Konrat et al. 1999), so it yields a single peak per residue, which provides better peak separation and also makes it possible to measure only the \(\textrm^_\textrm^_\) DD–\(\textrm_\textrm^}_\) DD rate. The pulse sequence is shown in Fig. 4. In this experiment, the coherence transfer starts as follows:

$$\begin &\textrm^\textrm_\xrightarrow []} 2\textrm^\textrm_\textrm_\mathrm}\xrightarrow [J_,J_]},^2J_}} 8\textrm^_\textrm'_\textrm_\mathrm}\textrm^_\quad\xrightarrow []}} \\ &\xrightarrow []}} 4\textrm'_\textrm_\mathrm}\textrm^_\Rightarrow \text \end$$

Fig. 4

Pulse sequence of the experiment 2 (\(\textrm^_\textrm^_\) DD–\(\textrm_\textrm^}_\) DD CCR). The evolution of \(^}\) is in constant-time mode during CCR evolution: \(A = (\textrm_+t_)/4\) and \(B = (\textrm_-t_)/4\), C\('\) evolution is in (semi)constant-time mode: \(a_2=(\varDelta _}+t_2)/2, \ b_2=t_2(1-\varDelta _})/(2t^_), \ c_2=\varDelta _}(1-t_2)/(2t^_)\) and N evolution is in constant-time mode. The semi-constant (Grzesiek and Bax 1993) or constant time mode is chosen automatically, based on the number of sampling points and spectral width in a given dimension. The delays were set as follows: \(\varDelta _}\) = 5.4 ms, \(\varDelta _}\) = 33.5 ms, \(\varDelta _}\) = 50 ms, \(\varDelta _}\) = 9.1 ms, and \(\varDelta _}\) = 3.4 ms. Total CCR rate evolution time is set as \(\textrm_\) = 28.6 ms. Unless noted explicitly, pulse phases are set to x. Phase \(\phi _\) depends on the version of the experiment: for reference experiment x, for transfer experiment y. The value of individual phase cycles are \(\phi _=x,-x\), \(\phi _=2(x),2(-x)\), \(\phi _=x\), \(\phi _=4(x),4(y),4(-x),4(-y)\), \(\psi =y\) and receiver \(\phi _=\phi _+\phi _+2\cdot \phi _\). Quadrature detection is achieved by States-TPPI (Marion et al. 1989) for \(^}\) (change of \(\phi _\) phase) and C\('\) (change of \(\phi _\) phase) evolutions, and echo-antiecho (Kay et al. 1992; Schleucher et al. 1993) for N evolution (change of \(\phi _\) and \(\psi\) phases and change of \(G_\) amplitude sign). Selective pulses for C\('\) and \(^}\) spins were Q3 (for 180\(^\circ\) pulses) and Q5 (for 90\(^\circ\) pulses) (Emsley and Bodenhausen 1992) The selective 180\(^\circ\) pulse on proton channel (marked \(_}\)) affects only \(^}\) and was prepared by Bruker WaveMaker tool, using offset = 4.3 ppm, bandwidth = 3 ppm, reburp shape (Geen and Freeman 1991). Simultaneous inversion of \(^}\) and C\('\) spins was achieved using 6-element composite pulse (Shaka 1985). The pulses labelled by a star (”*”) were executed only in the transfer version of the experiment. Rectangles represent hard pulses while rounded cones symbolize shaped pulses; the empty ones are 180\(^\circ\) pulses and the filled ones are 90\(^\circ\) pulses. Gradients with numbers from 1 to 12 are cleaning gradients. Gradients \(G_\) and \(G_\) are used for coherence selection in echo-antiecho quadrature detection. Proton decoupling is performed with composite pulse scheme waltz65 (Zhou et al. 2007) and nitrogen decoupling during acquisition is performed with composite pulse scheme garp (Shaka et al. 1985). The CCR and unifying blocks are indicated by rectangle boxes

The CCR block and the unifying block are identical as in Experiment 1 (\(\textrm^_\textrm^_\) DD–\(\textrm_\textrm^}_\) DD). Analogously, the \(\textrm_\textrm^\textrm_\) DD–\(\textrm^_\) CSA and \(\textrm_\) CSA–\(\textrm^_\) CSA were refocused during the CCR block, and \(\textrm^_\textrm^_\) DD–\(\textrm_\) CSA was not refocused, but did not lead to an observable magnetization (on the coherence transfer schemes below it is shown in squared brackets).

In the final fragment of the pulse sequence, we allow for the evolution of the chemical shifts of \(\textrm'_\)and \(\textrm_\). The evolution of the \(\textrm_\) chemical shift is executed in constant-time mode because the INEPT delay is very long (50 ms) and there is no need to implement a semi-constant time evolution option. The coherence transfer of this fragment of the pulse sequence is as follows:

$$\begin&4\textrm'_\textrm_\mathrm}\textrm^_+ [8\textrm^_\textrm'_\textrm^\textrm_\textrm_\mathrm}] \xrightarrow []}} \\&\quad \xrightarrow []}} 8\textrm^_\textrm'_\textrm_\mathrm}\textrm^_+ [4\textrm'_\textrm^\textrm_\textrm_\mathrm}] \xrightarrow [J_,J_]},^2J_}} \\&\quad \xrightarrow [J_,J_]},^2J_}} 2\textrm^\textrm_\textrm_\mathrm}+ [4\textrm^_\textrm_\mathrm}\textrm^_] \xrightarrow []} \\&\quad \xrightarrow []} \textrm^\textrm_+ [\mathrm] \end$$

Experiment 3. \(\textrm_\textrm^}_\) DD–\(\textrm_\textrm^}_\) DD

The third experiment measures the CCR effect between \(\textrm_\textrm^}_\) DD and \(\textrm_\textrm^}_\) DD. From the peak \(^}_)}}-\textrm_)}\) – \(\textrm'_\) – \(\textrm^\alpha _\)we get information on the \(\psi _)}\)and \(\phi _)}\) angles simultaneously. This CCR rate was previously measured in two ways: Pelupessy et al. (2003b) proposed a 2D quantitative gamma experiment (only nitrogen and proton evolution), and later Vögeli (2017) proposed 3D experiments (HN(CA)CON and HNCA(CO)N), where a difference in relaxation between zero-quantum and double-quantum coherences was evaluated and each peak appeared as a multiplet (which is not optimal for IDPs). The coherence transfer pathway of our experiment differs substantially from those previously proposed (see Fig. 5), starting from the excitation of \(\textrm^\) nuclei instead of \(\textrm^\textrm\) ones:

$$\begin & \textrm^_\xrightarrow []H^}} 2\textrm^_\textrm^_\xrightarrow [J_H^}]J_N},^J_N}}\\ & \rightarrow 4\textrm_\textrm^_\textrm_\mathrm}\Rightarrow \text \end$$

The CCR block is the same as in Pelupessy et al. (2003b).

Fig. 5

Pulse sequence of the experiment 3 (\(\textrm_\textrm^}_\) DD–\(\textrm_\textrm^}_\) DD CCR). The evolution of \(^}\) is in constant-time mode, C\('\) evolution is in (semi)constant-time mode: \(a_2=(\varDelta _}+t_2)/2, \ b_2=t_2(1-\varDelta _})/(2t^_), \ c_2=\varDelta _}(1-t_2)/(2t^_)\) and N evolution is in (semi)constant-time mode: \(a_3=(\varDelta _}+t_3)/2, \ b_3=t_3(1-\varDelta _})/(2t^_), \ c_3=\varDelta _}(1-t_3)/(2t^_)\). The semi-constant (Grzesiek and Bax 1993) or constant time mode is chosen automatically, based on the number of sampling points and spectral width in a given dimension. The delays were set as follows: \(A = \textrm_/4\), \(\textrm_\) = 28 ms, \(\varDelta _}\) = 5.4 ms, \(\varDelta _}\) = 31 ms, \(\varDelta _}\) = 29 ms, \(\varDelta _}\) = 55 ms, \(\varDelta _}\) = 9.1 ms, \(\varDelta _}\) = 9.4 ms, and \(\varDelta _}\) = 3.4 ms. Unless noted explicitly, pulse phases are set to x. Phase \(\phi _\) depends on the version of the experiment: for reference experiment x, for transfer experiment y. The value of individual phase cycles are \(\phi _=x,-x\), \(\phi _=2(x),2(-x)\), \(\phi _=x\), \(\phi _=4(x),4(y),4(-x),4(-y)\), \(\psi =y\) and receiver \(\phi _=\phi _+\phi _\). Quadrature detection is achieved by States-TPPI (Marion et al. 1989) for \(^}\) (change of \(\phi _\) phase) and C\('\) (change of \(\phi _\) phase) evolutions, and echo-antiecho (Kay et al. 1992; Schleucher et al. 1993) for N evolution (change of \(\phi _\) and \(\psi\) phases and change of \(G_\) amplitude sign). Selective pulses for C\('\) and \(^}\) spins were Q3 (for 180\(^\circ\) pulses) and Q5 (for 90\(^\circ\) pulses) (Emsley and Bodenhausen 1992). The pulses labelled by arrow ("\(\rightarrow\)") were shifted by \(\varDelta _}\)/4 to the right in the transfer version of the experiment. Rectangles represent hard pulses while rounded cones symbolize shaped pulses; the empty ones are 180\(^\circ\) pulses and the filled ones are 90\(^\circ\) pulses. Gradients with numbers from 1 to 12 are cleaning gradients. Gradients \(G_\) and \(G_\) are used for coherence selection in echo-antiecho quadrature detection. Proton decoupling is performed with composite pulse scheme waltz65 (Zhou et al. 2007) and nitrogen decoupling during acquisition is performed with composite pulse scheme garp (Shaka et al. 1985). The CCR and unifying blocks are indicated by rectangle boxes

The frequencies of the HA nuclei (which we excite at the beginning of the pulse sequence) are close to the frequency of water, therefore we add a purge pulse before the CCR block to crush the water magnetization.

During the CCR block, the CCR (\(\textrm_\textrm^}_\) DD–\(\textrm_\textrm^}_\) DD ) evolves, partially leading to the anti-phase magnetization of amide nitrogen nuclei with respect to amide protons. In the reference version of the experiment, the evolution of \(_\textrm}\) coupling is refocused, thus the ‘auto’ component in this version of the experiment has the form of in-phase magnetization. In the transfer version, the two 180\(^\circ\) proton pulses are shifted, leading to the evolution of \(_}}\) coupling. As a result, the ‘cross’ term in this version of the experiment has the form of in-phase magnetization (similarly to ‘auto’ component of the reference version). Therefore, no unifying block is employed in the pulse sequence. Three potentially significant unwanted CCR rates should be considered at this point. Evolution due to \(\textrm_\textrm^\textrm_\) DD–\(\textrm_\) CSA and \(\textrm_\textrm^\textrm_\) DD–\(\textrm_\) CSA was refocused in the CCR block by the 180\(^\circ\) pulses acting on amide proton nuclei. However \(\textrm_\) CSA–\(\textrm_\) CSA did evolve, but the resulting coherence was not transferred to an observable one (on the coherence transfer scheme below it is shown in squared brackets).

After the CCR block, on the way towards the observable amide proton magnetization, we evolve the chemical shifts of \(\textrm^\alpha _\), \(\textrm'_\) and \(\textrm_\), where only \(\textrm^\alpha _\) is measured in constant-time mode (the CT length of 55 ms provides not only a proper evolution of the scalar coupling of \(\textrm^\alpha _\)with nitrogen and carbonyl carbon, but also a minimization of the coupling with C\(^\beta\)). Here the final steps of the coherence transfer are shown:

$$\begin &4\textrm_\textrm^_\textrm_\mathrm}+ [16\textrm^\textrm_\textrm_\textrm^_\textrm^\textrm_\textrm_\mathrm}]\\&\quad\xrightarrow [J_C'}]J_N},^J_N}} 2\textrm^_\textrm'_\quad+\\&\quad+ [8\textrm^\textrm_\textrm^_\textrm'_\textrm^\textrm_] \xrightarrow [J_}]} \\&\quad\xrightarrow [J_}]} 2\textrm'_\textrm_\mathrm}\quad +\\&\quad+ [8\textrm^\textrm_\textrm'_\textrm_\mathrm}\textrm^\textrm_] \xrightarrow [}]} \\&\quad\xrightarrow [}]} 2\textrm^\textrm_\textrm_\mathrm}+ [2\textrm^\textrm_\textrm_\mathrm}]\xrightarrow []}\\&\quad\xrightarrow []} \textrm^\textrm_+ [ \mathrm] \end$$

Experiment 4. \(\textrm^\textrm_)}^_}\) DD–\(\textrm'_\) CSA

The fourth experiment measures the CCR effect between \(\textrm^\textrm_)}^_}\) DD and \(\textrm'_\) CSA. From the peak \(_)}-\textrm_)}}\) – \(\textrm'_\) – \(\textrm^\alpha _\) we get information on \(}\). This pulse sequence was published in 2020 (Kauffmann et al. 2020); please refer to this article for a full description.

Experiment 5. \(^\alpha _}\) \(^\alpha _}\) DD–\(\textrm'_\) CSA

The fifth experiment measures the CCR effect between \(\textrm^_\textrm^_\) DD and \(\textrm'_\) CSA. From the peak \(^}_)}}-\textrm_)}\) – \(\textrm'_\) – \(\textrm^\alpha _\) we get information on the \(}\) angle. The first measurement of this rate was made by Yang et al. (1997, 1998) in a 3D J-resolved HN(CO)CA experiment, where the CCR rates were calculated using the intensities of zero quantum/double quantum peaks. Later, Chiarparin et al. (1999) carried out a 2D quantitative gamma experiment (without carbon evolution) with shifting pulses in the CCR block, leading to an identical form of coherence at the end of the CCR block in both reference and transfer versions. Unlike these experiments, our experiment is not ‘out-and-back’, and the coherence transfer starts from \(^}\) rather than \(^\textrm}\) (see Fig. 6):

$$\begin \textrm^_\xrightarrow []H^}} 2\textrm^_\textrm^_\xrightarrow [J_H^}]C'}} 2\textrm^_\textrm'_\Rightarrow \text \end$$

Fig. 6