Christian Herbst

Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra

We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC′C and 3D C′NCA with sequential 13C acquisitions, 3D NHH and 3D NC′H with sequential 1H acquisitions and 3D CANH and 3D C’NH with broadband 13C–15N mixing are demonstrated using microcrystalline samples of the β1 immunoglobulin binding domain of protein G (GB1) and the chicken α-spectrin SH3 domain.

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.

Chemical shift correlation at high MAS frequencies employing low-power symmetry-based mixing schemes

An approach for conveniently implementing low-power CNnν and RNnν symmetry-based band-selective mixing sequences for generating homo- and heteronuclear chemical shift correlation NMR spectra of low γ nuclei in biological solids is demonstrated. Efficient magnetisation transfer characteristics are achieved by selecting appropriate symmetries requiring the application of basic RF elements of relatively long duration and numerically tailoring the RF field modulation profile of the basic element. The efficacy of the approach is experimentally shown by the acquisition of 15N–13C dipolar and 13C–13C scalar and dipolar coupling mediated chemical shift correlation spectra at representative MAS frequencies.

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

We have explored the design of broadband scalar coupling mediated 13C–13C and cross-relaxation suppressed 1H–1H TOCSY sequences employing phase/amplitude modulated inversion pulses. Considering a variety of supercycles, pulsewidths and a RF field strength of 10xa0kHz, the Fourier coefficients defining the amplitude and phase modulation profiles of the 180° 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 1H–1H 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.

Numerical design of RNnν symmetry-based RF pulse schemes for recoupling and decoupling of nuclear spin interactions at high MAS frequencies

An approach for the efficient implementation of RNnν symmetry-based pulse schemes that are often employed for recoupling and decoupling of nuclear spin interactions in biological solid state NMR investigations is demonstrated at high magic-angle spinning frequencies. RF pulse sequences belonging to the RNnν symmetry involve the repeated application of the pulse sandwich {RϕR−ϕ}, corresponding to a propagator URFxa0=xa0exp(−i4ϕIz), where ϕxa0=xa0πν/N and R is typically a pulse that rotates the nuclear spins through 180° about the x-axis. In this study, broadband, phase-modulated 180° pulses of constant amplitude were employed as the initial ‘R’ element and the phase-modulation profile of this ‘R’ element was numerically optimised for generating RNnν symmetry-based pulse schemes with satisfactory magnetisation transfer characteristics. At representative MAS frequencies, RF pulse sequences were implemented for achieving 13C–13C double-quantum dipolar recoupling and through bond scalar coupling mediated chemical shift correlation and evaluated via numerical simulations and experimental measurements. The results from these investigations are presented here.

An approach to NMR assignment of intrinsically disordered proteins.

An efficient approach to NMR assignments in intrinsically disordered proteins is presented, making use of the good dispersion of cross peaks observed in [(15) N,(13) C]- and [(13) C,(1) H(N) ]-correlation spectra. The method involves the simultaneous collection of {3D (H)NCO(CAN)H and 3D (HACA)CON(CA)HA} spectra for backbone assignments via sequential H(N) and H(α) correlations and {3D (H)NCO(CACS)HS and 3D (HS)CS(CA)CO(N)H} spectra for side-chain (1) H and (13) C assignments, employing sequential (1) H data acquisitions with direct detection of both the amide and aliphatic protons. The efficacy of the approach for obtaining resonance assignments with complete backbone and side-chain chemical shifts is demonstrated experimentally for the 61-residue [(13) C,(15) N]-labelled peptide of a voltage-gated potassium channel protein of the Kv1.4 channel subunit. The general applicability of the approach for the characterisation of moderately sized globular proteins is also demonstrated.

Recoupling and decoupling of nuclear spin interactions at high MAS frequencies: numerical design of CNnν symmetry-based RF pulse schemes

The CNnν 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 CNnν 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.

Broadband 15N–13C dipolar recoupling via symmetry-based RF pulse schemes at high MAS frequencies

An approach for generating efficient

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

{rm{RN}}_{n}^{nu_{rm{S}}, {nu_{rm{k}}}}