Sequential acquisition of multi-dimensional heteronuclear chemical shift correlation spectra with ¹H detection.

RF pulse schemes for the simultaneous acquisition of heteronuclear multi-dimensional chemical shift correlation spectra, such as {HA(CA)NH & HA(CACO)NH}, {HA(CA)NH & H(N)CAHA} and {H(N)CAHA & H(CC)NH}, that are commonly employed in the study of moderately-sized protein molecules, have been implemented using dual sequential (1)H acquisitions in the direct dimension. Such an approach is not only beneficial in terms of the reduction of experimental time as compared to data collection via two separate experiments but also facilitates the unambiguous sequential linking of the backbone amino acid residues. The potential of sequential (1)H data acquisition procedure in the study of RNA is also demonstrated here.

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

Two different NMR pulse schemes involving sequential (1)H data acquisition are presented for achieving protein backbone sequential resonance assignments: (i) acquisition of 3D {HCCNH and HNCACONH} and (ii) collection of 3D {HNCOCANH and HNCACONH} chemical shift correlation spectra using uniformly (13)C,(15)N labelled proteins. The sequential acquisition of these spectra reduces the overall experimental time by a factor of ≈2 as compared to individual acquisitions. The suitability of this approach is experimentally demonstrated for the C-terminal winged helix (WH) domain of the minichromosome maintenance (MCM) complex of Sulfolobus solfataricus.

Recoupling and decoupling of nuclear spin interactions at high MAS frequencies: numerical design of CN(n)(nu) symmetry-based RF pulse schemes.

The CN(n)(nu) class of RF pulse schemes, commonly employed for recoupling and decoupling of nuclear spin interactions in magic angle spinning solid state NMR studies of biological systems, involves the application of a basic "C" element corresponding to an RF cycle with unity propagator. In this study, the design of CN(n)(nu) symmetry-based RF pulse sequences for achieving 13C-13C double-quantum dipolar recoupling and through bond scalar coupling mediated 13C-13C chemical shift correlation has been examined at high MAS frequencies employing broadband, constant-amplitude, phase-modulated basic "C" elements. The basic elements were implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by an RF phase value. The phase-modulation profile of the "C" element was optimised numerically so as to generate efficient RF pulse sequences. The performances of the sequences were evaluated via numerical simulations and experimental measurements and are presented here.

Design of high-power, broadband 180 degrees pulses and mixing sequences for fast MAS solid state chemical shift correlation NMR spectroscopy.

An approach for the design of high-power, broadband 180 degrees pulses and mixing sequences for generating dipolar and scalar coupling mediated (13)C-(13)C chemical shift correlation spectra of isotopically labelled biological systems at fast magic-angle spinning frequencies without (1)H decoupling during mixing is presented. Considering RF field strengths in the range of 100-120 kHz, as typically available in MAS probes employed at high spinning speeds, and limited B (1) field inhomogeneities, the Fourier coefficients defining the phase modulation profile of the RF pulses were optimised numerically to obtain broadband inversion and refocussing pulses and mixing sequences. Experimental measurements were carried out to assess the performance characteristics of the mixing sequences reported here.

Heteronuclear J cross-polarisation in liquids using amplitude and phase modulated mixing sequences.

The design of mixing sequences for heteronuclear J cross-polarisation in the liquid state has been examined employing supercycles of amplitude/phase modulated RF pulses. The Fourier coefficients defining the modulation profiles of the pulses were optimised numerically so as to achieve efficient magnetisation transfer within the desired range of resonance offsets. A variety of supercycles, pulsewidths and RF field strengths were considered in implementing heteronuclear anisotropic and isotropic mixing sequences. The coherence transfer characteristics of the sequences obtained were evaluated by numerical simulations. The experimental performances of the sequences were tested by measurements carried out on a moderate sized protein at 750 MHz. The results presented demonstrate that the approach adopted in this study can be employed effectively to tailor, as per the experimental requirements and constraints, the RF-field modulation profiles of the pulses constituting the mixing scheme for generating heteronuclear J cross-polarisation sequences.

Broadband homonuclear TOCSY with amplitude and phase-modulated RF mixing schemes.

We have explored the design of broadband scalar coupling mediated (13)C-(13)C and cross-relaxation suppressed (1)H-(1)H TOCSY sequences employing phase/amplitude modulated inversion pulses. Considering a variety of supercycles, pulsewidths and a RF field strength of 10 kHz, the Fourier coefficients defining the amplitude and phase modulation profiles of the 180 degrees pulses were optimised numerically so as to obtain efficient magnetisation transfer within the desired range of resonance offsets. The coherence transfer characteristics of the mixing schemes were assessed via numerical simulations and experimental measurements and were compared with commonly used sequences based on rectangular RF pulses. The efficacies of the clean (1)H-(1)H TOCSY sequences were also examined via numerical simulations for application to weakly oriented systems and sequences with efficient, broadband and clean dipolar transfer characteristics were identified. In general, the amplitude and phase modulated TOCSY sequences presented here have moderately better performance characteristics than the sequences currently employed in biomolecular NMR spectroscopy.