Into the fold: Sensitivity-enhanced NMR

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  • Published: Oct 15, 2015
  • Author: David Bradley
  • Channels: NMR Knowledge Base
thumbnail image: Into the fold: Sensitivity-enhanced NMR

Sensitive and dynamic

Antiparallel beta-sheet structure of the enzyme catalase: The antiparallel hydrogen bonds (dotted) are between peptide NH and CO groups on adjacent strands. Arrows indicate the chain direction, and electron density contours outline the non-H atoms. Image: Wikimedia Commons (edited by MIT News)

Researchers at Massachusetts Institute of Technology have used sensitivity-enhanced dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) spectroscopy to analyse the structure that a yeast protein forms as it interacts with other proteins in a cell opening up new insights into protein folding and misfolding.

Biology is complicated. We can take that as read. At the protein level, there are a multitude of interactions and interactors, so unravelling the patterns and behaviour is difficult, especially when proteins may be present at low levels, interactions only fleeting and the matrix in which they occur contain many other proteins and biochemical reagents. Indeed, most techniques for studying proteins require much higher concentrations than the endogenous levels found naturally, several orders of magnitude higher, commonly. Those techniques also prefer purity. However, DNP can, according to the MIT team, "dramatically enhance the sensitivity" of NMR spectroscopy and allow structural studies in biologically matrices to be carried out at natural concentrations and in the natural, and complex, environment of the biological milieu. The biological context is particularly important for structural studies of proteins that have intrinsically disordered domains or environmentally sensitive folding pathways.  The yeast prion protein, Sup35, has both. Exogenously prepared carbon-13 isotopically labelled protein added to deuterated lysates and essentially makes the Sup35 visible in a biological environment that is “invisible” the team reports. Moreover, this facilitates DNP with great efficiency.

Standing out in a crowd

Proteins fold into their active forms in many different ways depending on the conditions and biochemical environment in which they find themselves. Understanding folding is important to protein scientists and others hoping to understand how proteins work. However, it is misfolding that is interesting from the biomedical perspective in that protein misfolding is commonly associated with degenerative diseases such as Alzheimer’s and Parkinson’s disease, and prion diseases, such as variant CJD (Creutzfeldt-Jakob disease). Observing the way in which a yeast prion behaves as it interacts with other proteins in a cell could lead to new clues as to how to understand, treat and perhaps one day even prevent protein misfolding diseases.

“Dynamic nuclear polarization has a capacity to transform our understanding of biological structures in their native contexts,” explains MIT biology professor Susan Lindquist. Robert Griffin, director of the Francis Bitter Magnet Laboratory, is co-lead author on the research published in the journal Cell, while former post-doctoral scientist Kendra Frederick, who is now at the University of Texas Southwestern, is the paper’s "lead" author.

To recap, conventional NMR exploits nuclear spin to reveal molecular structures. The chemical shifts can reveal the presence of an alpha helix or a beta sheet in the protein, for instance. However, conventional cabon-13 NMR is rather insensitive because it is a low gamma nuclei; even at high magnetic fields, only a tiny fraction of the carbon-13 atoms are polarized. Thus, for the last two decades Griffin’s research group has been developing DNP, which involves transferring polarization from unpaired electrons to protons and thence to carbon nuclei, using microwaves generated by a gyrotron, a high-frequency microwave oscillator developed in collaboration with Richard Temkin of MIT’s Department of Physics and Plasma Science and Fusion Center. This technique is coupled with the use of paramagnetic polarizing agents developed by chemist Tim Swager and his group at MIT. This combination allows the scientists to boost signal intensities in carbon-13 NMR spectra by a factors of 100 to 400, a rather significant increase in sensitivity.

The team reports that with standard solid-state NMR, they would need approximately 30 milligrams of purified protein to obtain sufficient useful information in a sufficiently short time. Conversely, with the DNP boost, they can work with unpurified protein at quantities that would normally be found inside a cell. To home in on only the protein of interest they label it with carbon-13 so that it stands out from the crowd.

Accessing new problems

“It’s opening up a completely new set of problems that we can access,” Frederick explains. “Using the sensitivity enhancement technique allows you to look at the protein at the correct levels, which is really important when you’re thinking about its biology.” This technique could open access to a wide range of protein studies in natural conditions seen in living things. Obviously, the proteins are not in crystalline form, which is required in X-ray diffraction and they do not need to be in a uniform solution.

Lindquist and colleagues have been studying Sup35 for many years. This protein usually functions to help cells terminate protein translation but in its prion form it exists as misfolded, tangled clumps known as amyloids that no longer work in this capacity. These are actually normal states for the yeast protein, one active the other inactive. However, in human, and other mammals, proteins that form amyloids are usually associated with diseases, in particular neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s and rheumatoid arthritis, and so their presence in otherwise healthy tissue is highly undesirable.

In earlier work, using standard NMR with purified Sup35, researchers demonstrated that a large section of the protein, which forms the amyloid, has the pleated, beta-sheet structure, while a separate, large section is just disordered and flops around in the cell with no consistent structure. The DNP NMR shows that this purportedly disordered region is anything but floppy. In the natural environment of the protein, surrounded by other cellular proteins, the intrinsically disordered region adopts a regular structure akin to the beta sheet. Higher resolution spectra are now needed to determine the atomic level details of this conformational change..

Frederick plans to continue using this NMR technique to study other yeast proteins, as well as human amyloid proteins. In particular, she told SpectroscopyNOW, she wants "to study how cellular environments influence the folding pathways of meta-stable and intrinsically disordered proteins. In addition to prion biology in yeast, she plans on investigating structures of toxic and non-toxic conformations of the proteins involved in protein-folding based neurodegenerative diseases such as Alzheimer's, Parkinsons' and Huntingtin's.  This work will tightly couple cellular genotypes and phenotypes to protein structure."

Related Links

Cell 2015, online: "Sensitivity-Enhanced NMR Reveals Alterations in Protein Structure by Cellular Milieus"

Article by David Bradley

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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