Differentially labeled flaviviral protease-cofactor complex for NMR spectroscopic applications

Abstract

Flaviviruses such as Dengue, Zika and West-Nile viruses have a positive strand RNA genome which is translated to a polyprotein inside the host cell. The viral polypeptide is matured to its constituents by the enzymatic action of NS2B-NS3 protease-cofactor complex. The flaviviral protease-cofactor complex attracted a lot of interest recently because of its potential for therapeutic intervention and the unique nature of catalysis where the peptide cofactor regulates the enzymatic activity. Obtaining the enzyme and cofactor differentially labeled with naturally abundant nuclei and NMR active nuclei respectively will be helpful in reducing the spectral complexity by making the enzyme invisible in a multidimensional NMR spectrum while only showing peaks from the cofactor. This will enable one to study the properties of the cofactor in isolation using NMR spectroscopy. Here, I have used a strategy for selectively labeling the cofactor within the complex with NMR active nuclei while peaks from the enzyme were rendered invisible. The protocol used here takes advantage of an ‘on-column unfolding’ step during the initial Ni-NTA chromatography to separate the enzyme and cofactor in unfolded conditions. The labeled cofactor was then allowed to fold in the presence of an unlabeled enzyme to obtain a differently labeled complex. We compared the ¹H-¹⁵N HSQC spectrum of the differently labeled, wild type and free cofactor to ensure that the cofactor attained the desired fold within the complex. The protocol is scalable, inexpensive and can be applied to other two-component enzyme systems where a peptide cofactor is essential for the folding of an enzyme.