3D-printed microcell for protein NMR at high ionic strengths and small sample volumes.

Abstract

Standard solution NMR measurements use 5 mm outer diameter (OD) sample tubes that require ca. 0.5 mL of solvent to minimize "end effects" on magnetic field homogeneity in the active volume of the sample. Shigemi cells reduce the solvent requirement to ca. 0.29 mL. At high ionic strength or at ultrahigh magnetic fields, smaller OD samples are needed to study samples in conductive, radiofrequency-absorbing solvents such as water. We demonstrate an effective and inexpensive alternative for reducing the active sample volume to 0.13 mL by 3D printing ellipsoidal shaped cells that are inserted into 5 mm OD NMR tubes. Static magnetic susceptibility, $\chi$ , of printer resin was measured using a simple slice-selection pulse sequence. We found that the $\chi$ of water increases linearly with NaCl concentration from $-9.05$ to $-8.65$ ppm for 0 to 2 M NaCl. The $\chi$ of D₂O was measured to be $-9.01$ ppm. The susceptibility difference between the resin ( $\chi =-9.40$ ppm) and water can be minimized by paramagnetic doping of the resin. Such doping was found to be unnecessary for obtaining high-quality protein NMR spectra when using ellipsoidal-shaped cells that are insensitive to susceptibility mismatching. The microcells offer outstanding radiofrequency (RF) and good $B_o$ homogeneities. Integrated 600 MHz heteronuclear single quantum coherence (HSQC) signal intensities for the microcell sample in phosphate-buffered saline (PBS) buffer were $6.5\pm 4$ % lower than for 0.5 mL of the same protein solution in a regular 5 mm sample tube. The cell is demonstrated for N-acetylated $\alpha$ -synuclein in PBS buffer and for observing tetramerization of melittin at 2 M NaCl.