NMR spectroscopy enhanced with amplifier

Researchers typically determine the structure and dynamics of proteins using NMR (nuclear magnetic resonance) spectroscopy; until now, however, much higher concentrations were necessary for in vitro measurements of the biomolecules in solution than found in our body’s cells. An NMR method enhanced by a powerful amplifier, in combination with molecular dynamics simulation, can now enable their detection and characterisation at physiological concentrations.

Currently, NMR spectroscopy is the only method that allows a complete description of the atomic structure of biomacromolecules in their native solution state. However, due to the inherently low sensitivity of the method, the samples must contain many more molecules per volume than are physiologically common. To overcome this discrepancy, hyperpolarisation (more precisely, dissolution dynamic nuclear polarisation) can be used to achieve a 1000-fold signal amplification in NMR measurements.

“Spectroscopy bears some similarities with an electric guitar,” said Dennis Kurzbach, from the Institute of Biological Chemistry at the University of Vienna. “If the amplifier is too weak, you will hear very little if you do not hit the strings strongly, meaning that you need a lot of material to see an NMR signal. With the new hyperpolarisation amplifier, you can now see something even at low concentration.”

Kurzbach and colleagues set out to measure biomolecules at concentrations as low as 1 µmol/L, approaching that of our cells. This is important because proteins can react to unnaturally high concentrations, in that they no longer do what they are supposed to do and suddenly behave differently.

A dissolution dynamic nuclear polarisation measurement typically provides one-dimensional spectra, which limits the obtained information. To describe proteins comprehensively under natural concentration conditions, the researchers employed molecular dynamics simulations. “We were also able to extrapolate the fingerprint we obtained of our molecule via NMR to its ‘entire body’, ie, its multidimensional structure,” Kurzbach said.

The value of this methodological advance is demonstrated using the ubiquitous transcription factor MAX. This protein can self-associate with various other proteins (ie, protein dimerisation). For example, MYC-MAX dimers have a great influence on the DNA copying processes in the cell.

With the new methods, MAX has been shown to adopt an undocumented conformation when concentrations approach physiological levels. According to Kurzbach, “The folding spectrum of MAX is of crucial importance for working together with MYC and thus for the proliferation of healthy as well as diseased cells in the body.”

The new method can thus help to better understand the process of cell proliferation to tumour growth and thus elucidate basic mechanisms for cancer development. This is just one of many potential applications for the method, which has been published in the journal Science Advances — after all, thousands of proteins in our cells perform a wide variety of tasks, including digestion and regulation of DNA and RNA.

Image caption: Co-author Dennis Kurzbach and first author Ludovica M Epasto in front of the hyperpolarisation device. Image credit: D Kurzbach, L Epasto.

Please follow us and share on Twitter and Facebook. You can also subscribe for FREE to our weekly newsletters and bimonthly magazine.