Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations

Longhini, Andrew P.; LeBlanc, Regan M.; Becette, Owen; Salguero, Carolina; Wunderlich, Christoph H.; Johnson, Bruce A.; D'Souza, Victoria M.; Kreutz, Christoph; Dayie, T. Kwaku

The first advantage of these new site-specific labels is the potential for new assignment schemes. We have coupled alternate labeling of either C1 or C2 labeled ribose to C8 labeled purine bases. These combinations have allowed us to develop a new NOESY assignment strategy that benefits from reduced spectral crowding. This new strategy takes advantage of the large proton chemical shift differences between the C1 and C2 ribose carbons. By using an alternating C1 and C2 pattern with labeled bases, the NOESY spectrum is greatly simplified without compromising the information content present. Since all nucleotides are labeled, a complete NOESY walk is possible in helical regions. Additionally if the purines and pyrimidines labeled with C1 and C2 enrichment are reversed, orthogonal data is generated that can confirm the previous assignment.