NMR Spectroscopy

NMR spectroscopy can provide 3D structures of proteins, and additionally infromation on proteins at atomic resolution, for example protein dynamics and pKa active sites. Protein structures in living cells can be studied by non-invasive NMR spectroscopy.

When studying molecules with disordered regions, NMR spectroscopy as a research method has an advantage to x-ray crystallography: disordered regions complicate crystallization and using NMR as the study method alleviates this problem, since NMR requires no crystallization. Still, even in NMR analysis, disordered regions produce signal overlap. 

Segmental isotopic labelling

One way to facilitate the NMR analysis is segmental isotopic labeling which enables the analysis to be focused on just one domain of an entire protein. This also keeps the studied protein as close as possible to its actual structure. Segmental isotopic labeling can be achieved by a variety of methods, but most unfortunately leave a “scar” which may affect the protein. Iwai lab is researching techniques to perform segmental isotopic labeling that leaves no scar. 

Another problem with producing segmentally isotopic labeled samples for NMR analysis by split Inteins is their poor solubility, and we have done experiments on extremophile-originated inteins, which possess a higher level of solubility. 

When studying a protein, in structural biology it has been a common practice to remove the disordered regions from the globular domain, since aforementioned complications to crystallization. However, this dissection may influence the conformation of the studied domain. (Ciragan et al, 2020)

We are interested in examining proteins as whole constructs, and work on overcoming the hurdles regarding low-complexity regions (LCCR) in proteins and their effect on NMR analysis. Furthermore, intrinsically disordered proteins, or proteins with disordered regions have recently been recognized to be an important part of biological functions, such as allosteric regulation and complex formation. When LCCRs are taken into account, signal assignments can become even more time-consuming and difficult, since the degeneracy of NMR signals becomes more prevalent.