LoperThorne814

The Protein Data Bank tracks the number of structures solved of all types of biomolecule (not just proteins as the name implies but also nucleic acids, but most are proteins). The vast majority of the proteins have been solved by Crystallography using, in most cases, high energy synchrotron sources; the key to solving a crystal structure is growing a crystal. That is relatively straight forward to do nowadays for most soluble proteins. For membrane proteins, it is exceedingly difficult. Only a couple of hundred out of the 50,000 total structures in the PDB are membrane proteins and for proteins involved in neurodegeneration, so called amyloid proteins, they essentially cannot be studied by these methods. Solution NMR is the other major competitor for x-ray crystallography. More than 5,000 protein structures have been solved that way. It is a powerful tool for addressing soluble proteins with the molecular weight of less than 20,000 Daltons or so. In some instances it has been applied to larger proteins. But the limitation is that one must find the conditions where the protein is soluble in aqueous buffer. To study membrane proteins by solution NMR requires solublisation in detergents and this is often an impediment to finding good sample conditions. So in solid state NMR we can avoid a lot of those complications because we don't need the sample to be soluble in order to crystallise it or to study it in solution NMR where it also has to be soluble. So solid state NMR is uniquely able to address those types of proteins that are not soluble and do not form crystals.

Tell us about the solid state NMR developed in your lab and how it works.

First of all, we prepare the protein using 13C and 15N growth media. We prepare them from bacterial expressions vectors in E. coli and that enriches the 13C and 15N nuclei in the protein to improve the sensitivity of the spectra. Then we perform magic angle spinning which is a technique to improve the resolution of the spectra. Essentially what magic angle spinning does is it gives high resolution spectra despite the fact that the sample was not tumbling in aqueous solution. Normally, in solution NMR, the molecule must tumble rapidly in the magnetic field in order to give high resolution spectra. With magic angle sinning we can avoid that requirement. Rather than relying on the tumbling of the molecule in solution, we essentially perform that averaging process for the molecule by rotating the sample very rapidly within the magnetic field.