[et_pb_section fb_built=”1″ _builder_version=”4.23″ _module_preset=”default” custom_padding=”5px|||||” global_colors_info=”{}”][et_pb_row _builder_version=”4.23″ _module_preset=”default” border_width_bottom=”1px” border_color_bottom=”#e2e2e2″ global_colors_info=”{}”][et_pb_column type=”4_4″ _builder_version=”4.23″ _module_preset=”default” global_colors_info=”{}”][et_pb_post_title meta=”off” featured_image=”off” _builder_version=”4.23″ _module_preset=”default” global_colors_info=”{}”][\/et_pb_post_title][\/et_pb_column][\/et_pb_row][et_pb_row use_custom_gutter=”on” gutter_width=”2″ _builder_version=”4.23″ _module_preset=”default” global_colors_info=”{}”][et_pb_column type=”4_4″ _builder_version=”4.23″ _module_preset=”default” global_colors_info=”{}”][et_pb_text _builder_version=”4.23″ _module_preset=”default” custom_padding=”0px|||||” global_colors_info=”{}”]<\/p>\n
Solid-state NMR has had considerable success in the structure determination of perdeuterated proteins.\u00a0<\/span><\/span>However, one would like to use a fully protonated protein samples for various reasons.\u00a0we have developed new 1<\/sup>H-1<\/sup>H recoupling sequences that selectively recouple protons in the solid-state. In fully protonated molecules, SERP\u00a0can be used to measure quantitative 1<\/sup>H-1<\/sup>H distances while BASS-SD is used to observe 1<\/sup>H-1<\/sup>H structural restraints similar to those observed only in perdeuterated proteins with protons at selective sites. <\/span><\/p>\n <\/span><\/p>\n <\/span><\/p>\n The <\/span>CN\u00a0<\/i>symmetry sequences,<\/span>routinely used in solid-state NMR, rely on the rotation properties of spatial and spin tensors.Symmetry sequences are used to efficiently recouple any anisotropic interaction via a suitable choice of N, n and nu<\/span>\u00a0symmetry numbers.\u00a0We modified the sequences to\u00a0evolves the isotropic chemical shift during recoupling while the symmetry numbers ensure that only the DQ dipolar terms are symmetry allowed in the first-order Hamiltonian.\u00a0Similar to SERP, the dipolar Hamiltonian is modulated by isotropic chemical shifts. Selective recoupling for a spin pair is achieved by setting the transmitter offset at the center of the two proton resonances.\u00a0More than 30 homonuclear CNn<\/sub>v<\/sup><\/i>sequences were evaluated through simulations in terms of the scaling factor, selectivity, and polarization transfer efficiency.\u00a0A major advantage of symmetery sequence over SERP is that rf requirements are only 50% of the MAS frequency and transfer efficiencies are two to three-fold higher. Selective C64<\/sub>1<\/sup>has a narrower bandwidth than SERP recoupling and therefore is more selective than SERP sequences. These sequences were also applied for the measurement of 1<\/sup>H-1<\/sup>H contacts in fully protonated samples. The influence of the other proton makes it challenging to determine 1<\/sup>H-1<\/sup>H distances using the modified C64<\/sub>1<\/sup>sequence.\u00a0<\/span><\/p>\n<\/div>\n [\/et_pb_text][et_pb_image src=”https:\/\/www.tifrh.res.in\/~vipin\/wp-content\/uploads\/2024\/08\/Screenshot-from-2024-08-06-21-54-13.png” _builder_version=”4.23″ _module_preset=”default” title_text=”Screenshot from 2024-08-06 21-54-13″ align=”center” hover_enabled=”0″ sticky_enabled=”0″][\/et_pb_image][et_pb_text _builder_version=”4.23″ _module_preset=”default” global_colors_info=”{}”]<\/p>\n SERP <\/span>\u00a0is a phase-modulated recoupling sequence <\/span>to <\/span><\/span>selectively and quantitatively measure magnetic dipole-dipole interaction between protons at fast MAS. The recoupling conditions of numerically optimized SERP have been explained in <\/span><\/span>the framework of the bimodal Floquet theory with two characteristic frequencies spinning frequency and the modulation frequency of the pulse sequence.<\/span><\/span>17-20<\/span><\/sup>\u00a0The effective recoupled Hamiltonian consists of isotropic chemical shift and <\/span><\/span>DQ dipolar-coupling terms. This is a time-dependent Hamiltonian and is not suitable for recoupling. Selective recoupling between two proton spins is achieved by setting the transmitter offset to the center of the two proton-resonances, thereby ensuring that the isotropic chemical shift term for the two protons is eliminated. Under this condition, only the dipolar coupling term between the two protons is time-independent and can be used to measure the1<\/sup>H-1<\/sup>H dipole coupling. In a two-spin system, SERP recoupling has an efficiency of ~53%<\/span><\/p>\n [\/et_pb_text][et_pb_image src=”https:\/\/www.tifrh.res.in\/~vipin\/wp-content\/uploads\/2024\/08\/Screenshot-from-2024-08-06-21-54-47.png” title_text=”Screenshot from 2024-08-06 21-54-47″ align=”center” _builder_version=”4.23″ _module_preset=”default” hover_enabled=”0″ global_colors_info=”{}” sticky_enabled=”0″][\/et_pb_image][et_pb_image src=”https:\/\/www-cxcp1.tifrh.res.in\/~vipin\/wp-content\/uploads\/2023\/11\/Overlay_H_alpha_H_methyl_004.png” title_text=”Overlay_H_alpha_H_methyl_004″ align=”center” _builder_version=”4.23″ _module_preset=”default” global_colors_info=”{}”][\/et_pb_image][et_pb_text _builder_version=”4.23″ _module_preset=”default” global_colors_info=”{}”]<\/p>\n Transfer of magnetization during SERP recoupling from HNA46\u2192 H\u03b1F45\/A46. The HN A46 distances to the two H\u03b1 are similar and the H\u03b1 chemical shift is overlapped. A simulation (HNA46\u2192 H\u03b1F45\/A46, black solid line) was performed with the distances obtained from X-ray structure and plotted with a similar build up obtained from experiments (filled circles). The maxima were artificially scaled to overlap. Spectra obtained from SERP mixing at two different conditions on Ubiquitin<\/p>\n<\/div>\n This project has been in collaboration with Dr. Yusuke Nishiyama of JEOL-Japan. <\/em><\/p>\n <\/em><\/p>\n Band Selective Spectral Spin-Diffusion (BASS-SD)\u00a0<\/span><\/i><\/b>is a method to obtain selective1<\/sup>H-1<\/sup>H contacts between chemically similar protons such as HN<\/sup>-HN<\/sup>\/H<\/span>\u03b1<\/span><\/sup>-H\u03b1<\/sup>\/Hmethyl<\/sup>-Hmethyl<\/sup>in the range of 5-6\u00c5 <\/span>despite the presence of other protons at shorter distances in fully protonated proteins.\u00a0<\/span><\/span>In fully protonated samples, BASS-SD gives correlations similar to those observed in perdeuterated samples with protons located at selective sites. BASS-SD recoupling combines low-power spin-lock pulse<\/span>\u00a0on protons with fast MAS to enable the selective exchange of polarization amongst protons (e.g.<\/span>\u00a0selective HN<\/sup>-HN<\/sup>transfer). <\/span>Bimodal Floquet analysis was used to understand the mechanism of polarization transfer. The analysis shows that second-order cross-terms between two homonuclear dipolar couplings mediate polarization transfer during BASS-SD recoupling. Simultaneously, the isotropic chemical shifts in the first-order Hamiltonian provide the selectivity <\/span>. The selectivity is essential to observe selective <\/span>HN<\/sup>-HN<\/sup>\/H<\/span>\u03b1<\/span><\/sup>-H\u03b1<\/sup>\/Hmethyl<\/sup>-Hmethyl<\/sup>correlation.<\/span><\/p>\n [\/et_pb_text][et_pb_image src=”https:\/\/www.tifrh.res.in\/~vipin\/wp-content\/uploads\/2024\/08\/Figure2_005-768×1213-1.png” title_text=”Figure2_005-768×1213″ align=”center” _builder_version=”4.23″ _module_preset=”default” hover_enabled=”0″ global_colors_info=”{}” sticky_enabled=”0″][\/et_pb_image][et_pb_text _builder_version=”4.23″ _module_preset=”default” global_colors_info=”{}”]<\/p>\n Figure a) pulse sequence for the 3D (H)NHH experiment b) Overlay of 2D (H)NH (Blue) and 2D (H)N(H)H-BASS-SD (Red) spectra. c-d) HN-HN restraints from 3D (H)N(H)(H)NH spectra of fully protonated U-[13C,15N]-GB1 at 111.11 kHz MAS with c) BASS-SD (red, 5 ms) and d) RFDR ( black, 1 ms) mixing. Observed HN-HN correlations across the e) \u03b2-sheet and f) \u03b1-helix motifs are mapped on the X-ray structure of GB1 (PDB code: 2QMT). Cyan and grey spheres denote amide and other protons, respectively, while the dark gray and blues sticks represent the backbone atoms. 2D 15N-15N positive projections from the 3D spectra are shown in g) for BASS-SD and h) RFDR, i) 15N- filtered 1D proton intensity comparison after RFDR and BASS-SD mixing for different experiments.<\/p>\n<\/div>\nSelective1<\/sup>H-1<\/sup>H recoupling<\/span>based onCNn<\/sub>v<\/sup><\/span>sequences<\/span><\/h4>\n
Selective Recoupling of Protons (SERP)<\/span><\/h4>\n
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Relevant Publications:<\/h4>\n
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