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  • Title: Real-time pure shift ¹⁵N HSQC of proteins: a real improvement in resolution and sensitivity.
    Author: Kiraly P, Adams RW, Paudel L, Foroozandeh M, Aguilar JA, Timári I, Cliff MJ, Nilsson M, Sándor P, Batta G, Waltho JP, Kövér KE, Morris GA.
    Journal: J Biomol NMR; 2015 May; 62(1):43-52. PubMed ID: 25737243.
    Abstract:
    Spectral resolution in proton NMR spectroscopy is reduced by the splitting of resonances into multiplets due to the effect of homonuclear scalar couplings. Although these effects are often hidden in protein NMR spectroscopy by low digital resolution and routine apodization, behind the scenes homonuclear scalar couplings increase spectral overcrowding. The possibilities for biomolecular NMR offered by new pure shift NMR methods are illustrated here. Both resolution and sensitivity are improved, without any increase in experiment time. In these experiments, free induction decays are collected in short bursts of data acquisition, with durations short on the timescale of J-evolution, interspersed with suitable refocusing elements. The net effect is real-time (t 2) broadband homodecoupling, suppressing the multiplet structure caused by proton-proton interactions. The key feature of the refocusing elements is that they discriminate between the resonances of active (observed) and passive (coupling partner) spins. This can be achieved either by using band-selective refocusing or by the BIRD element, in both cases accompanied by a nonselective 180° proton pulse. The latter method selects the active spins based on their one-bond heteronuclear J-coupling to (15)N, while the former selects a region of the (1)H spectrum. Several novel pure shift experiments are presented, and the improvements in resolution and sensitivity they provide are evaluated for representative samples: the N-terminal domain of PGK; ubiquitin; and two mutants of the small antifungal protein PAF. These new experiments, delivering improved sensitivity and resolution, have the potential to replace the current standard HSQC experiments.
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