Partial 13C isotopic enrichment of nucleoside monophosphates: useful reporters for NMR structural studies

Abstract

Analysis of the 13C isotopic labeling patterns of nucleoside monophosphates (NMPs) extracted from Escherichia coli grown in a mixture of C-1 and C-2 glucose is presented. By comparing our results to previous observations on amino acids grown in similar media, we have been able to rationalize the labeling pattern based on the well-known biochemistry of nucleotide biosynthesis. Except for a few notable absences of label (C4 in purines and C3′ in ribose) and one highly enriched site (C1′ in ribose), most carbons are randomly enriched at a low level (an average of 13%). These sparsely labeled NMPs give less complex NMR spectra than their fully isotopically labeled analogs due to the elimination of most 13C–13C scalar couplings. The spectral simplicity is particularly advantageous when working in ordered systems, as illustrated with guanosine diphosphate (GDP) bound to ADP ribosylation factor 1 (ARF1) aligned in a liquid crystalline medium. In this system, the absence of scalar couplings and additional long-range dipolar couplings significantly enhances signal to noise and resolution.