Heteronuclear detection cryogenic probe head (Bruker TXO)

This proposal requests a new Nuclear Magnetic Resonance (NMR) hardware accessory, a TXO cryoprobe, to observe proteins with detail that is not available by other techniques. The addition of a TXO cryoprobe would give a competitive advantage to our NMR facility in an increasingly important research technology.

Proteins are essential biomolecules that are required for the structure, function, and regulation of the body's tissues and organs. A detailed understanding of how proteins work is essential to explain many biological processes, particularly in the context of disease. A protein is a chain of amino acids; many can fold into a defined shape that dictates its function, and accidental changes in the building blocks - the amino acids - can change the shape or flexibility of a protein to prevent it from functioning properly.

Proteins are not rigid and static; many can flex or change shape as part of how they work. It is necessary for us to also look at their flexibility (dynamics) to understand how they function. One of the methods to study shape and interactions in atomic detail is NMR spectroscopy.

NMR can look at proteins in solution, with conditions matched as closely as possible to those experienced in their natural physiological context. By observing proteins in solution NMR can not only look at their three-dimensional (3D) shape, but it can detect dynamic motion in different parts of a protein. In addition, NMR looks at an ensemble of molecules at the same time, sometimes identifying multiple alternative shapes that might be relevant for drug discovery.

Until recently, it was assumed that functioning protein chains must be folded into a compact 3D structure. However, a growing awareness of proteins with no preferred structure is challenging this concept. Intrinsically disordered proteins (IDPs), or proteins with a substantial intrinsically disordered region (IDR), are important for signalling and regulation of cellular functions.

NMR is uniquely suited to study IDPs, and as such methods have been developed to enhance its capabilities to do so. The basic components of a NMR spectrometer are a superconducting magnet, and a radio transmitter/receiver system connected to a probe head with sensor coils surrounding the actual sample volume - centred within the magnet. In a typical NMR experiment, radio wave bursts excite NMR-active nuclei of atoms such as hydrogen, carbon, and nitrogen and the receiver coil senses the responses from one element.

Not all isotopes can be detected by NMR and protein samples require labelling with NMR-active isotopes of carbon (13C) and nitrogen (15N). A separate signal received for each individual atom informs on the local environment and dynamic properties at each site, and is analysed to reveal 3D shapes or how mobile certain regions of a protein are. For folded proteins, the most informative and sensitive nuclei are hydrogen (1H) - that is why most NMR spectrometers are optimised for 1H detection, with the receiver coil for 1H closest to the sample.

For IDPs and IDRs, the local environment for individual hydrogen atoms is so similar that we have great difficulty distinguishing their signals from each other, impeding our examination of shape, dynamics or molecular interactions. To overcome this we can change the experiment to detect signal from 13C or 15N atoms instead. Specifically, carbonyl 13C signals from the backbone of each amino acid are much better resolved than the signals from 1H.

However, to do so most effectively requires a probe head which has the 13C/15N receiver coil closest to the sample, optimised for detecting 13C (or 15N) with high sensitivity. The proposed TXO 13C/15N-detect cryoprobe allows us to apply 13C-detect methods to a broader range of protein or nucleic acid research targets than we can address using our TCI probes that are optimised for 1H-detection.