Real-time pure shift 15N HSQC of proteins: a real improvement in resolution and sensitivity

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 (t2) 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 15N, while the former selects a region of the 1H 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-015-9913-z) contains supplementary material, which is available to authorized users.

Practical aspects of real‐time pure shift HSQC experiments

“…In principle, the quality of CTP enforcement can be improved by varying the amplitudes of the gradient pulse pairs in successive cycles of the acquisition loop, but in practice, the unmatched pulse pairs within two cycles suffice, as shown in Figure . The use of triple axis gradients, where available, is a more general way to prevent accidental refocusing …”

“…Provided the chunk duration and τ jr are small compared to T 2 , the effect of the extra relaxation during τ jr is just to increase the relaxation contribution to the measured linewidth by a factor (1 + τ jr /τ ch ) to (1 + τ jr /τ ch )/(π T 2 ), in hertz . In practice, for small molecules, the impact of the extra broadening is often small given the relatively short total acquisition times commonly used in HSQC, but in systems such as proteins that have short T 2 s, and where τ is relatively long, as for example dictated by the 1 J NH coupling constant in 15 N HSQC or by long selective pulses in Zangger–Sterk or band‐selective experiments, the extra line broadening can significantly degrade the resolution obtainable …”

“…Although the sidebands are very small when the echo time is short, which was the case in the first publication of real‐time HSQC, using gradient pulses lengthens the echo time and results in very strong periodic artefacts—see Figure (d, iii). The use of a modified BIRD element, in which a second carbon inversion pulse follows the BIRD element, is essential to refocus heteronuclear J evolution during the τ 2 periods . The original BIRD element and many of its variants, including the modified version implemented here, have been discussed in detail by Uhrín et al The duration of this second carbon pulse needs to be compensated for if the chemical shift evolution of the active spins during the J ‐refocusing element (τ jr period) is to be perfectly refocused.…”

“…Where strong gradient pulse pairs are employed during acquisition, we have noted a small (ppb) shift of all F 2 resonance frequencies attributable to disturbance of the magnetic field and/or the field‐frequency lock system . Inverting the signs of all gradient pulses changes the sign of the frequency error, and its amplitude is proportional to the strength of the gradient pulses applied.…”

“…Thus, instead of a pure shift interferogram being constructed from a series of chunks of data acquired in separate experiments, a pure shift FID is acquired in a continuous series of chunks of data interspersed with J ‐refocusing sequence elements. Real‐time acquisition using a BIRD J ‐refocusing element was first proposed by Lupulescu et al and has been used inter alia for 1D proton measurements and for HSQC experiments using BIRD and/or band‐selective methods …”