Exploring the limits of solution-state NMR in application to challenging proteins in drug discovery.
Russell Raj Pandian Senthamarai.
Date of Issue2012
School of Biological Sciences
Solution state Nuclear Magnetic Resonance Spectroscopy, despite its emergence as an important technique has its own limits: rapid transverse relaxation proportional to the molecular size of the protein, slow longitudinal relaxation and resonance overlap with increasing molecular size. While the limitations have been addressed consistently the quantitative limits of solution NMR experiments remain unclear. In the present work, we reported a unified framework, which enables a uniform benchmarking of all the available solution NMR experiments in terms of their sensitivity and tolerance to transverse relaxation. Ability to predict the sensitivity per unit time across dimensions has also been demonstrated and validated. Subsequently, an online NMR-DataBase has been developed accumulating the most commonly used solution NMR experiments. A backbone resonance assignment of DENV4 NS3 helicase, a 51 kDa protein, has been discussed. Less than 10% of the resonance assignment has been achieved with triple resonance. With the introduction of selectively labeled amino acids, another 4 residues have been assigned, portraying the necessity of alternative approaches such as PRE/PCS.