Structure-function relationship in an archaebacterial methionine sulphoxide reductase B.

Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be 'repaired' by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine. Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs.

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

An approach for generating efficient NR(vS, vk)(n) symmetry-based dual channel RF pulse schemes for gamma-encoded broadband (15)N-(13)C dipolar recoupling at high magic angle spinning frequencies is presented. The method involves the numerical optimisation of the RF phase-modulation profile of the basic "R" element so as to obtain heteronuclear double quantum dipolar recoupling sequences with satisfactory magnetisation transfer characteristics. The basic "R" element was implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by a RF phase and amplitude values. The performance characteristics of the sequences were evaluated via numerical simulations and (15)N-(13)C chemical shift correlation experiments. Employing such (13)C-(15)N double-quantum recoupling sequences and the multiple receiver capabilities available in the current generation of NMR spectrometers, the possibility to simultaneously acquire 3D NCC and CNH chemical shift correlation spectra is also demonstrated.

Solution structure of a functional biomimetic and mechanistic implications for nickel superoxide dismutases.

The nickel complex of a synthetic nonapeptide (HCDLPCFVY-NH2) is capable of catalytically disproportionating O2(.-) and is thus a functional biomimetic for nickel superoxide dismutases. This represents a simplification as compared to a NiSOD "maquette" that is based on a dodecapeptide that was recently reported [Inorg. Chem. 2006, 45, 2358]. The 3D solution structure reveals that the first six residues form a stable macrocyclic structure with a preformed binding site for Ni(II). Proline 5 exhibits a trans peptide linkage in the biomimetic and a cis conformation in NiSOD enzymes. DFT calculations reveal the source of this preference. Mechanistic consequences for the mode of action (identity of the fifth ligand) are discussed. The SOD activity is compared to enzymatic systems, and selected modifications allowed the biomimetic to be reduced to a functional minimal motif of only six amino acids (ACAAPC-NH2).

1H, 13C and 15N resonance assignment of a zinc-binding methionine sulfoxide reductase type-B from the thermophilic archeabacterium Methanothermobacter thermoautotrophicus.

The (1)H, (13)C and (15)N resonance assignment of a type-B zinc-binding methionine sulfoxide reductase lacking a 'recycling' cysteine from the thermophilic archeabacterium Methanothermobacter thermoautotrophicus is reported.

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.