Sequential protein NMR assignments in the liquid state via sequential data acquisition

Modern Technologies of Solution Nuclear Magnetic Resonance Spectroscopy for Three-dimensional Structure Determination of Proteins Open Avenues for Life Scientists

“…Furthermore, advanced techniques for protein sample preparation (e.g., SAIL technology [42], [174], [175], [176], [177], methyl group-selective 1 H, 13 C-labeling with deuteration of the other non-labile protons [178], [179], [180], [181], [182], [183]) combined with elaborate NMR pulse schemes (e.g., rapid NMR data collection such as the Band Selective Optimized-Flip-Angle Short-Transient (SOFAST), Band-selective Excitation Short-Transient (BEST) [184], [185] and their easy set up/use scripts (http://www.ibs.fr/research/scientific-output/software/pulse-sequence-tools/), ASAP or ALSOFAST [186] methods, the 3D or 4D methyl-methyl NOESY based on high-resolution and diagonal-free HMQC-NOESY-HMQC pulse schemes [181], [187], [188], [189], and the dual- or parallel-FID acquisition approaches [190], [191], [192]), and non-uniform data sampling (NUS) applying NMR data collection of the indirect dimension and quantitative reconstitution of NMR spectra from sparsely sampled data [193], [194], [195], should facilitate the study of challenging proteins by NMR (e.g., membrane proteins, enzymes such as kinase/phosphatase, and supramolecular complexes). Therefore, solution NMR spectroscopy is expected to develop even further as a tool for determining the tertiary structure of macromolecules and large molecular weight protein complexes (ca.…”

A Set of Efficient nD NMR Protocols for Resonance Assignments of Intrinsically Disordered Proteins

“…Even the availability of the additional RN[N‐CA HEHAHA]CAHA data set would not only provide further confirmation of the sequential connectivities established via the RN[N‐CA HEHAHA]NH protocol but also be potentially useful in establishing reliable sequential connectivities in regions where the amide proton signal intensities are inherently weak due to exchange of the labile protons with water. Representative spectral cross‐sections taken from 3D HN[N‐CA HEHAHA](CACS)HS and HN[N‐CA HEHAHA](N)H data sets collected using the sequential data acquisition procedure, without sparse sampling in the indirect dimensions and without and with the application of 13 C‐ 13 C TOCSY mixing are given in the SI. It is worth pointing out that, unlike the INEPT procedure, the application of band‐selective 15 N‐ 13 C α HEHAHA mixing sequence for transferring the 15 N i magnetisation to the adjacent “ i +1” and “ i −1” residues makes it possible to conveniently extract the side chain chemical shift information simultaneously.…”

“…Some of the possibilities to extract these,s imultaneously with the connectivity data, are outlined below.O ne possibility would be to adapt the RN[N-CAH EHAHA]NH protocols to achieve the simultaneous collection of RN[N-CAH EHAHA]-CACSHSc orrelation spectra.T his could be achieved by subjecting the residual 13 C a (i/iÀ1) magnetisation presenta tt he end of the HEHAHA mixing period to ap eriod of 13 C- 13 CT OCSY mixing first and then transferring the resultant 13 C sc magnetisation to the attached side chain protons througha 13 C-1 Hc ross polarization step for detection as in our earlier studies. [5] Even the availability of the additional RN[N-CAH EHAHA]CAHA data set would not only providef urtherconfirmation of the sequential connectivities established via the RN[N-CAH EHAHA]NH protocolb ut also be potentially useful in establishing reliable sequential connectivitiesi nr egions where the amide proton signal intensities are inherently weak due to exchange of the labile protons with water.R epresentative spectral cross-sections taken from 3D HN[N-CA HEHAHA](CACS)HS and HN[N-CA HEHAHA](N)H data sets collected using the sequential dataa cquisitionprocedure, [5,60] withoutsparse sampling in the indirect dimensions and without andw ith the application of 13 C-13 C TOCSY mixinga re given in the SI. It is worth pointingo ut that, unlike the INEPT procedure, the application of band-selective 15 N-13 C a HEHAHA mixing sequence for transferring the 15 N i magnetisation to the adjacent "i + 1" and" iÀ1" residues makes it possible to conveniently extract the side chain chemical shift information simultaneously.T he pulse sequences can also be adapted, using dual receiversa si ne arlier studies [61][62][63][64] to acquire simultaneously RN[N-CAH EHAHA]NCO and RN[N-CA HE-HAHA]CAHA correlation data sets.…”

UTOPIA NMR: activating unexploited magnetization using interleaved low-gamma detection

“…In line with other techniques (Kupce et al 2010; Parella and Nolis 2010; Perez-Trujillo et al 2007; Salzmann et al 2000; Wiedemann et al 2014), our UTOPIA setup relies on the acquisition of several spectra using one single relaxation recycle period. Considering that in most NMR experiments the vast amount of time required to acquire a multidimensional spectrum is used up by this recycle period (i.e.…”