Efficient and stable solid nuclear magnetic resonance heteronuclear correlation spectrum method

Abstract

The invention discloses an efficient and stable solid nuclear magnetic resonance heteronuclear correlation spectrum method, and belongs to the technical field of nuclear magnetic resonance. The method comprises the following steps: instrument configuration and sample preparation; 90-degree pulse widths of three channels of 1H, 13C and 15N are measured, and corresponding channel parameters are set according to the 90-degree pulse widths; and performing a heteronuclear correlation experiment to obtain a heteronuclear correlation spectrum. According to the invention, spatial connection information among 1H, 13C and 15N sites can be observed, the efficiency of 13C-15N heteronuclear polarization transfer is remarkably improved, and the experimental stability is good.

Description

Technical Field

[0001] The present invention relates to a solid nuclear magnetic resonance (NMR) experimental method, in particular to a method based on 1 Protein solid-state NMR heteronuclear correlation spectroscopy method for H detection. Background Art

[0002] Nuclear Magnetic Resonance (NMR), as a non-destructive testing method, has been widely used in many fields such as chemistry, biology, medicine and materials science. Solid-state NMR obtains high-resolution NMR spectra by introducing magic-angle spinning (MAS), and reintroduces the dipole coupling eliminated by MAS through dipole recoupling technology, thereby revealing the spatial relationship between atomic nuclei. Solid-state NMR has become a key analytical tool for studying the structure and dynamics of solid-state materials, and is particularly suitable for the study of biological macromolecules such as membrane proteins and drugs. Compared with crystal diffraction, liquid NMR and cryo-electron microscopy, solid-state NMR can study the true structure and dynamics of proteins in an environment close to physiological conditions, showing unique advantages.

[0003] The solid-state NMR spectra of proteins are usually very complex and require multidimensional solid-state NMR technology to disperse the signals for subsequent data analysis. 13 C- 15 N heteronuclear correlation can provide information about the connections within and between amino acids in proteins, which is crucial for accurate protein analysis. 13 C- 15 N is mainly established using the cross polarization (CP) method. This method has good performance at low and medium MAS speeds (≤40 kHz), but its efficiency gradually decreases with the increase of MAS speed, and becomes inefficient and unstable at ultra-high speeds (≥100 kHz), which ultimately makes biological solid NMR experiments at ultra-high speeds very time-consuming.

[0004] In view of this, proposing an efficient and stable solid NMR heteronuclear correlation method to improve the efficiency of biological solid NMR experiments at ultra-high speeds has become a key issue that needs to be urgently solved by technical personnel in this field. Summary of the invention

[0005] The present invention provides an efficient and stable solid nuclear magnetic resonance heteronuclear correlation spectroscopy method, which can improve the efficiency of biological solid NMR experiments at ultra-high speeds, shorten the time of multidimensional correlation experiments, and is very stable, reducing the requirements for the stability of the instrument's radio frequency field and the precise setting of the experiment.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: Heterokaryotic correlation experiments 13 C- 15 N polarization transfer uses 13 C- 15 N heteronuclear dipole recoupling method. 13 C- 15 The N heteronuclear dipole recoupling method is 13 C and 15 The recoupling module with specific pulse units is repeatedly used in N channels. The characteristics of the recoupling module are: each recoupling module contains 16 pairs of simultaneously transmitted pulses with different phases. 13 C pulse unit P1P2P3 and 15 N pulse units P1P2P3, and the duration of a single recoupling module is 36 magic angle rotation periods τ R .

[0007] The specific experimental steps are as follows: Debug the spectrometer, prepare the sample to be tested, place the rotor containing the sample to be tested into the sample chamber of the probe, rotate and tune.

[0008] Collect one dimension 1 H spectrum, set 1 H center frequency, determination 1 H channel 90° pulse width.

[0009] optimization 1 H- 13 C Cross-polarization parameters, maximize the peak height or area, and measure 13 C channel 90° pulse width, calibration sample 13 The central chemical shift value of the C spectrum.

[0010] optimization 1 H- 15 N is the cross-polarization parameter that maximizes the peak height or area. 15 N channel 90° pulse width, calibration sample 15 The central chemical shift value of the N spectrum.

[0011] Experiments were performed using the hCNH3D pulse sequence, with the following settings: 1 H. 13 C and 15 The frequency center of N is set 1 H- 15 N cross polarization, 1 H- 13 C cross-polarization parameters, 1 H. 13 C. 15N channel 90° pulse and other parameters; set 1 H decoupling parameter, so 1 H RF field strength (i.e. 1 H Larmor precession frequency) is 15 kHz; set 1 H water peak suppression parameter, 1 H radio frequency field strength is 10 kHz; In the hCNH3D experiment, 13 C- 15 N polarization transfer uses 13 C- 15 N heteronuclear dipole recoupling method: According to 13 C. 15 Calculation of 90° pulse width of N channel for heteronuclear dipole recoupling 13 C and 15 N RF field strength, 13 The RF field strength of the C channel (i.e. 13 C Larmor precession frequency) is equal to the magic angle rotation frequency (ν R ), so that 15 The RF field strength of the N channel (i.e. 15 N Larmor precession frequency) is equal to n times the magic angle rotation frequency, optimizing the recoupling time to maximize the spectral peak height or area.

[0012] Set the number of accumulations, loop delay, indirect dimension increment and number of points, and perform Fourier transform after sampling to obtain the multidimensional correlation spectrum.

[0013] The above method is optional. 13 C- 15 N heteronuclear dipole recoupling method 15 N-channel and 13 The pulses of the C channel can be interchanged.

[0014] The above method is optional. 13 C- 15 In the N heteronuclear dipole recoupling method, the value of n, which represents the radio frequency field intensity in the two channels, is 0.55, and its variation range can vary between 0.35 and 0.75.

[0015] The above method is optional. 13 C- 15 N heteronuclear dipole recoupling method can be used to 13 C to 15 N polarization transfer can also be used 15 N to 13 Polarization transfer of C.

[0016] The above method is optional. 13 C- 15The N heteronuclear dipole recoupling method was carried out under high-speed magic angle spinning conditions greater than 40 kHz.

[0017] The above method is optional. 13 C- 15 N heteronuclear dipole recoupling method 13 C core or 15 The N nucleus can be replaced by other spin nuclei with a spin quantum number of 1 / 2.

[0018] The present invention has the following advantages and positive effects: 1) Different 1 H. 13 C. 15 Spatial proximity or chemical bond connection information of N; 2) 13 C- 15 The N heteronuclear dipole recoupling method does not require very detailed optimization and can easily achieve high efficiency. 15 N- 13 The efficiency improvement of the CCP method can reach 70%.

[0019] 3) 13 C- 15 The N heteronuclear dipole recoupling method has good stability and can carry out long-term multidimensional experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0021] Figure 1 The present invention provides 13 C- 15 N- 1 Schematic diagram of the hCNH3D pulse sequence for H correlation experiments.

[0022] Figure 2 The present invention provides 13 C- 15 Schematic diagram of the pulse sequence of the N heteronuclear dipole recoupling method.

[0023] Figure 3 It is Fmoc-U- 13 C, 15 Two-dimensional analysis of the N-labeled Met-Leu-Phe (fMLF) tripeptide 13 Cα-( 15 N)-1 H correlation spectrum.

[0024] Figure 4 It is Fmoc-U- 13 C, 15 2D N-labeled Met-Leu-Phe (fMLF) tripeptide 13 Cα)- 15 N- 1 H correlation spectrum.

[0025] Figure 5 It is Fmoc-U- 13 C, 15 Two-dimensional analysis of the N-labeled Met-Leu-Phe (fMLF) tripeptide 13 C'-( 15 N)- 1 H correlation spectrum.

[0026] Figure 6 It is Fmoc-U- 13 C, 15 2D N-labeled Met-Leu-Phe (fMLF) tripeptide 13 C')- 15 N- 1 H correlation spectrum. Implementation

[0027] In order to facilitate the understanding and implementation of the present invention by those skilled in the art, the present invention is described in detail in a clear and complete manner in conjunction with the drawings in the embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0028] The present invention is an innovation based on the solid NMR heteronuclear correlation spectrum experimental method based on hydrogen detection. 13 C- 15 N- 1 H or 15 N- 13 C- 1 In the H multidimensional correlation experimental method, 13 C- 15 N-polarization transfer is mainly achieved through cross polarization (CP), which has problems such as low efficiency and poor stability. 13 C- 15 N-polarized transmission provides an efficient and stable 13 C- 15 N- 1H multidimensional correlation experiment. It is worth mentioning that the experimental method in the present invention has wide applicability. Most solid materials containing hydrogen and other atoms with nuclear spin quantum numbers of 1 / 2 can be used to conduct efficient and stable heteronuclear correlation experiments through this method for the analysis of complex spectra. In addition, the heteronuclear dipole recoupling method in the present invention is also applicable to other related experiments.

[0029] See also Figure 1 and Figure 2 As shown, the present invention discloses an efficient and stable solid nuclear magnetic resonance heteronuclear correlation spectroscopy method. Figure 1 The present invention provides 13 C- 15 N- 1 Schematic diagram of the hCNH3D pulse sequence for H correlation experiments, which shows the initial 1 H magnetization direction 13 C, 13 C evolved 13 C- 15 N heteronuclear dipole recoupling method 15 N, 15 N evolved 15 N- 1 H cross polarization 1 H, last 1 H detection. φ1, φ2, φ3, φ4, φ5, φ6, φ7 are the phase cycles of the pulses, φ R is the phase cycle of the receiver. These phase cycles only represent one of the phase cycles and can be adjusted in practice according to the coherent transfer path of the specific experiment.

[0030] Figure 2 The present invention provides 13 C- 15 Schematic diagram of the pulse sequence of the N heteronuclear dipole recoupling method, which shows that 13 C and 15 In N channels, pulse units P1P2P3 (P1, P2, P3 represent three pulses) with specific RF field power and specific phase are used simultaneously and repeatedly to achieve 13 C- 15 N heteronuclear polarization transfer. 13 C- 15 A single recoupling module of the N heteronuclear dipole recoupling method contains 16 pairs of 13 C and 15 The total duration of the N-channel P1P2P3 pulse unit is 36 magic angle rotation periods τ RThe basic phase of the pulse unit P1P2P3 is (0゜, 120゜, 0゜); (x, y, -x, -y) corresponds to (0゜, 90゜, 180゜, 270゜), which is the phase supercycle of the pulse; the superposition of the basic phase and the phase supercycle constitutes the final phase of each pulse. Embodiment 1

[0031] based on Figure 1 and Figure 2 The implementation of the solid-state NMR heteronuclear correlation spectroscopy method comprises the following steps: Debug the spectrometer, prepare the sample to be tested, put the rotor with the sample to be tested into the sample chamber of the probe, rotate and tune; Collect one dimension 1 H spectrum, set 1 H center frequency, determination 1 H channel 90° pulse width; optimization 1 H- 13 C Cross-polarization parameters, maximize the peak height or area, and measure 13 C channel 90° pulse width, calibration sample 13 The central chemical shift value of the C spectrum; optimization 1 H- 15 N is the cross-polarization parameter that maximizes the peak height or area. 15 N channel 90° pulse width, calibration sample 15 The central chemical shift value of the N spectrum; Experiments were performed using the hCNH3D pulse sequence, with the following settings: 1 H. 13 C and 15 The frequency center of N is set 1 H- 15 N cross-polarization parameters, 1 H- 13 C cross-polarization parameters, 1 H. 13 C. 15 N channel 90° pulse parameters; setting 1 H decoupling parameter, so 1 H RF field strength is 15 kHz; set 1 H water peak suppression parameter, 1 H radio frequency field strength is 10 kHz; In the hCNH3D experiment, 13 C- 15 N polarization transfer uses 13 C- 15 N heteronuclear dipole recoupling method: According to 13 C. 15Calculation of 90° pulse width of N channel for heteronuclear dipole recoupling 13 C and 15 N RF field strength, 13 The RF field strength of channel C is equal to the magic angle spinning frequency ν R n times, so 15 The radio frequency field intensity of the N channels is equal to n times the magic angle rotation frequency, and the recoupling time is optimized to maximize the spectrum peak height or area; the number of accumulations, loop delay, indirect dimension increment and number of points are set, and Fourier transform is performed after sampling to obtain a multidimensional correlation spectrum.

[0032] Specifically, 13 C- 15 A recoupling module of the N heteronuclear dipole recoupling method contains 36 magic angle rotation periods τ R .

[0033] Specifically, 13 C- 15 A recoupling module of the N heteronuclear dipole recoupling method contains 16 pairs of 13 C and 15 N pulse units P1P2P3 (P1, P2, P3 represent three pulses) emitted simultaneously by two channels.

[0034] Specifically, 13 C- 15 The recoupling module of the N heteronuclear dipole recoupling method is repeated until the recoupling module ends.

[0035] Specifically, 13 C- 15 N heteronuclear dipole recoupling method 15 N-channel and 13 The pulses of the C channel can be interchanged.

[0036] Specifically, 13 C- 15 In the N heteronuclear dipole recoupling method, the value of n, which represents the radio frequency field intensity in the two channels, is 0.55, and its variation range can vary between 0.35 and 0.75.

[0037] Specifically, 13 C- 15 N heteronuclear dipole recoupling method can be used to 13 C to 15 N polarization transfer can also be used 15 N to 13 Polarization transfer of C.

[0038] Specifically, 13 C- 15 The N heteronuclear dipole recoupling method was carried out under high-speed magic angle spinning conditions greater than 40 kHz.

[0039] Specifically, 13 C- 15 N heteronuclear dipole recoupling method 13 C core or 15 The N nucleus can be replaced by other spin nuclei with a spin quantum number of 1 / 2. Embodiment 2

[0040] In a specific embodiment, see Figure 1 and Figure 2 As shown, measure and set 1 H. 13 C. 15 N 90° pulse, optimized and set 1 H- 13 C CP, 1 H- 15 N CP, water pressure peak and heteronuclear decoupling conditions, optimized and set 13 C- 15 N heteronuclear dipole recoupling method, setting the t1, t2, and t3 parameters for chemical shift evolution.

[0041] The experimental conditions of this example are: Bruker 700 MHz spectrometer, 0.7 mm diameter rotor, magic angle rotation frequency 100 kHz, cycle delay 1 s, accumulation times 16 times. The sample is Fmoc-U- 13 C, 15 N-labeled Met-Leu-Phe (fMLF) tripeptide powder, used Figure 1 and Figure 2 The pulse sequence is shown.

[0042] The specific steps include: Debug the spectrometer, prepare the fMLF sample to be tested, put the rotor with the fMLF sample into the sample chamber of the probe, rotate it to 100 kHz, and then tune it; One-dimensional sampling 1 H spectrum, 1 The H center frequency was set at 8.0 ppm and the 1 The 90° pulse width of the H channel is 0.75μs; optimization 1 H- 13 C is the cross-polarization parameter, 13 C center frequency is set at 54.0 ppm (for 13 Cα) or 172.0 ppm (for carbonyl carbon 13 C'), so that the peak height or area is maximized, and the 13 The C channel 90° pulse width is 2.26 μs; optimization 1 H-15 N is the cross-polarization parameter, 15 The center frequency of N is set at 118.0 ppm to maximize the peak height or area. 15 The 90° pulse width of the N channel is 2.84 μs. 15 The central chemical shift of the N spectrum; Use the hCNH3D pulse sequence and set 1 H. 13 C and 15 The frequency center of N is set 1 H- 15 N cross polarization, 1 H- 13 C cross-polarization parameters, 1 H. 13 C. 15 N channel 90° pulse and other parameters; set 1 H decoupling parameter, so 1 H RF field strength is 15 kHz; set 1 H water peak suppression parameter, 1 H radio frequency field strength is 10 kHz; set up 13 C- 15 N heteronuclear dipole recoupling method module, based on the measured 13 C. 15 Calculation of heteronuclear dipole recoupling by N-channel 90° pulse width 13 C and 15 N RF field strength, 13 The RF field strength of channel C is equal to 55 kHz, making 15 The RF field strength of channel N is equal to 55 kHz, and the pulse units P1P2P3 are set to P1 = 74.25°, P2 = 297°, and P3 = 74.25°. 15 N- 13 Cα polarization transfer and 15 N- 13 C' transfer, the optimized recoupling times are 3.96 ms and 5.4 ms respectively; Set the number of accumulations to 16 and the loop delay to 1 s: (1) For two-dimensional 13 Cα-( 15 N)- 1 H correlation spectrum ( Figure 3 ), fix t2 to 0, 13 The increment and number of points of the C indirect dimension are 125 μs and 160 points respectively. After sampling, Fourier transform is performed to obtain the correlation spectrum; (2) For the two-dimensional ( 13 Cα)- 15 N- 1 H correlation spectrum ( Figure 4 ), fix t1 to 0, 15 The N-dimensional increment and number of points are 250 μs and 160 points respectively. After sampling, Fourier transform is performed to obtain the correlation spectrum; (3) For two-dimensional 13 C'-( 15 N)- 1 H correlation spectrum ( Figure 5 ), fix t2 to 0, 13 The increment and number of points of the C indirect dimension are 125 μs and 160 points respectively. After sampling, Fourier transform is performed to obtain the correlation spectrum; (4) For the two-dimensional ( 13 C')- 15 N- 1 H correlation spectrum ( Figure 6 ), fix t1 to 0, 15 The increment and number of points in the N indirect dimension are 250 μs and 160 points, respectively. After sampling, Fourier transform is performed to obtain the correlation spectrum.

[0043] The above steps do not represent the only order between the steps, but only represent one of the orders. And some steps may be repeated to adjust the optimal parameters according to the specific experiment.

[0044] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. An efficient and stable solid-state nuclear magnetic resonance heteronuclear correlation spectroscopy method, characterized in that: Heterokaryotic correlation experiments 13 C- 15 N polarization transfer uses 13 C- 15 N heteronuclear dipole recoupling method. 13 C- 15 The N heteronuclear dipole recoupling method is 13 C and 15 The recoupling module with specific pulse units is repeatedly used in N channels. The characteristics of the recoupling module are: each recoupling module contains 16 pairs of simultaneously transmitted and different phases 13 C pulse unit P1P2P3 and 15 N pulse units P1P2P3, and the duration of a single recoupling module is 36 magic angle rotation periods τ R .

2. The efficient and stable solid-state nuclear magnetic resonance heteronuclear correlation spectroscopy method according to claim 1, characterized in that: The following steps are also included: Debug the spectrometer, prepare the sample to be tested, put the rotor with the sample to be tested into the sample chamber of the probe, rotate and tune; Collect one dimension 1 H spectrum, set 1 H center frequency, determination 1 H channel 90° pulse width; optimization 1 H- 13 C Cross-polarization parameters, maximize the peak height or area, and measure 13 C channel 90° pulse width, calibration sample 13 The central chemical shift value of the C spectrum; optimization 1 H- 15 N is the cross-polarization parameter that maximizes the peak height or area. 15 N channel 90° pulse width, calibration sample 15 The central chemical shift value of the N spectrum; Experiments were performed using the hCNH3D pulse sequence, with the following settings: 1 H. 13 C and 15 The frequency center of N is set 1 H- 15 N cross-polarization parameters, 1 H- 13 C cross-polarization parameters, 1 H. 13 C. 15 N channel 90° pulse parameters; set 1 H decoupling parameter, so 1 H RF field strength is 15 kHz; set 1 H water peak suppression parameter, 1 H radio frequency field strength is 10 kHz; In the hCNH3D experiment, 13 C- 15 N polarization transfer uses 13 C- 15 N heteronuclear dipole recoupling method: According to 13 C. 15 Calculation of 90° pulse width of N channel for heteronuclear dipole recoupling 13 C and 15 N RF field strength, 13 The RF field strength of channel C is equal to the magic angle spinning frequency ν R n times, so 15 The radio frequency field intensity of the N channels is equal to n times the magic angle rotation frequency, and the recoupling time is optimized to maximize the spectrum peak height or area; the number of accumulations, loop delay, indirect dimension increment and number of points are set, and Fourier transform is performed after sampling to obtain a multidimensional correlation spectrum.

3. The efficient and stable solid-state nuclear magnetic resonance heteronuclear correlation spectroscopy method according to claim 1, characterized in that: 13 C- 15 N heteronuclear dipole recoupling method 13 C channel and 15 The pulses in the N channels can be interchanged.

4. The efficient and stable solid-state nuclear magnetic resonance heteronuclear correlation spectroscopy method according to claim 1, characterized in that: 13 C- 15 N heteronuclear dipole recoupling method 13 C channel and 15 The value of n representing the radio frequency field strength in the N channel is 0.55, and its variation range can vary between 0.35-0.

75.

5. The efficient and stable solid-state nuclear magnetic resonance heteronuclear correlation spectroscopy method according to claim 1, characterized in that: 13 C- 15 N heteronuclear dipole recoupling method for 13 C to 15 N polarization transfer, also used for 15 N to 13 Polarization transfer of C.

6. The efficient and stable solid-state nuclear magnetic resonance heteronuclear correlation spectroscopy method according to claim 1, characterized in that: 13 C- 15 The N heteronuclear dipole recoupling method was carried out under high-speed magic angle spinning conditions greater than 40 kHz.

7. The efficient and stable solid-state nuclear magnetic resonance heteronuclear correlation spectroscopy method according to claim 1, characterized in that: 13 C- 15 N heteronuclear dipole recoupling method 13 C core or 15 The N nucleus can be replaced by other spin nuclei with a spin quantum number of 1 / 2.

Images