New NMR experiments for nucleic acids and intrinsically disordered proteins

The aim of the project was to utilize the advanced high dimensional NMR methods with NUS implementations for the chemical shift assignments in the spectra of intrinsically disordered proteins/protein regions and nucleic acids. The project also involved the development of new high dimensional approaches for nucleic acids. Intrinsically disordered proteins are in abundance in organisms and their study is essential for understanding numerous diseases, in particular cancer and neurodegenerative disorders. Since they lack a stable structure and have fast conformational dynamics, their NMR spectra are characterized by low chemical shift dispersion as compared to well-structured globular proteins, which leads to severe peak overlap and makes NMR studies of large IDPs a challenging task. The goal of the project was to implement already developed NUS techniques for the analysis of a number of proteins samples. High dimensional experiments (4D, 5D) were acquired for achieving complete resonance assignments. Besides, spectral assignments for various proteins, this research work also included at the development of new high dimensional (4D) NMR experiments for structural studies of nucleic acids, in particular, ribonucleic acids (RNAs). Compared to well-structured globular proteins, NMR spectra of nucleic acids are severely overlapped which makes their structural studies a challenging task. This research work was an attempt to alleviate aforementioned issues by meticulously developing new methods especially suited for large RNAs. Apart from new methodology, the proposed experiments provided increased spectral dispersion and enhanced resolution which are the crucial advantages over conventional NMR methods used for nucleic acids. The developed experiments, 4D HC(P)CH and 4D HPCH provided (a) alternate methods with improved resolution and dispersion; (b) unambiguous resonance assignments for sequential analysis and (c) high quality restrains for structural calculations of nucleic acids. Apart from structure determination, the new experiments also complemented contemporary methods and tools for better characterization of conformational properties of RNAs.