Ultra-High Sensitivity in High-Resolution Relaxometry by Using a Cryo-probe and a Fast Mechanical Sample Shuttle Device

Field-dependent relaxation has been an active field of research for decades. However, its application to the investigation of the site-specific dynamics of macromolecular systems was restricted due to the lack of suitable hardware. In 2003, Redfield designed a sample-shuttle system and installed it on a high field solution state NMR spectrometer (1, 2). Applications to protein dynamics were benefited by such mechanical (2, 5) as well as pneumatic sample-shuttle hardware (4) and showed insights into nanosecond dynamics. We have recently designed a high speed mechanical shuttle device with high precision and stability and without compromise in sensitivity, except for the loss of signal due to relaxation decay. The device was installed on a conventional high resolution 600 MHz spectrometer with a 5 mm QXI probehead without modification within 30 min at Academia Sinica, Taiwan (3). The shuttling time is 80 ms for a distance of 90 cm from the center of the magnet at 14.1 T to the top of the magnet at 0.01 T. This permits us to determine relaxation times down to the order of 0.08 s in a routine basis with protein samples at 1 mM in a conventional 5 mm NMR tube. We now demonstrate that the same design can be installed and operated safely in a 700 MHz spectrometer equipped with a 5 mm QCI cryoprobe at CEA Saclay, France.

Recent field cycling applications in proteins were studied at concentration equal and even higher than 1 mM (4, 5) because of signal sensitivity issues. However, many proteins have to be studied at concentrations in the low hundreds of μM in order to avoid aggregation and unexpected polymerization. Here we demonstrate the enhanced signal sensitivity of the "field-cycler" on 700 MHz spectrometer equipped with 5 mm cryoprobe on 600 μM 15N labeled ubiquitin under high spectral resolution conditions. The sensitivity comparison of the field cycler working on the two standard-bore NMR systems experimentally was found to agree with theoretical calculation. The median signal to noise ratio of field cycling spectrum collected on 700 MHz with the cryoprobe and shuttled at 5 T with relaxation delay 0.1 s is 90.9 in the experimental condition of 8 scans in TD1682(1H) x 128(15N). The shuttle operation on the this system displays high stability.

We demonstrate that the field-cycler can be installed on various types of NMR spectrometers, including systems equipped with a cryoprobe to benefit of the full sensitivity of state-of-the-art biomolecular NMR systems. This set up provides unprecedented high sensitivity and spectral resolution in field cycling experiments, extending measurements of field-dependent relaxation on most proteins amenable to conventional NMR studies, including low concentration proteins with low solubility and disordered proteins.