Keeping PASE with WEFT: SHWEFT–PASE pulse sequences for 1H NMR spectra of highly paramagnetic molecules

Abstract

Metalloproteins are a category of biomolecules in which the metal site is usually the locus of activity or function. In many cases, the metal ions are paramagnetic or have accessible paramagnetic states, many of which can be studied using NMR spectroscopy. Extracting useful information from (1)H NMR spectra of highly paramagnetic proteins can be difficult because the paramagnetism leads to large resonance shifts (~400 ppm), extremely broad lines, extreme baseline nonlinearity, and peak shape distortion. It is demonstrated that employing polychromatic and adiabatic shaped pulses in simple pulse sequences, then combining existing sequences, leads to significant spectral improvement for highly paramagnetic proteins. These sequences employ existing technology, with available hardware, and are of short duration to accommodate short nuclear T1 and T2. They are shown to display uniform excitation over large spectral widths (~75 kHz), accommodate high repetition rates, produce flat baselines over 75 kHz while maintaining peak shape fidelity, and can be used to reduce spectral dynamic range. High-spin (S = 5/2) metmyoglobin, a prototypical highly paramagnetic protein, was used as the test molecule. The resulting one-dimensional (1D) pulse sequences combine shaped pulses with super-water elimination Fourier transform, which can be further combined with paramagnetic spectroscopy to give shaped pulses with super-water elimination Fourier transform-paramagnetic spectroscopy. These sequences require, at most, direct current offset correction and minimal phasing. The performance of these sequences in simple (1)H 1D, 1D NOE, and two-dimensional NOESY experiments is demonstrated for metmyoglobin and Paracoccus denitrificans Co(2+)-amicyanin (S = 3/2), and employed to make new heme hyperfine resonance assignments for high-spin metBjFixLH(151-256), the heme sensing domain of Bradyrhizobium japonicum FixL.