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.

Role of helical constraints of the EBS1–IBS1 duplex of a group II intron on demarcation of the 5′ splice site

“…We compared and plotted chemical shift difference between NMPs (Jezowska- Trzebiatowska et al 1980;Kishore et al 2005;Eichhorn et al 2012;Kwan 2012) and ID3. The chemical shift difference follows δ (NMP)-δ (ID3) convention, to obtain positive values for differences in δ, because NMPs display mostly downfield shifts relative to those of structured residues.…”

Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR

“…Here again the ratio of the sum of the intensities of satellite peaks to the sum of the intensities of the satellite and center peaks was used to calculate the absolute enrichment level. Third, for those carbon sites where no quantitative information could be obtained from either the 1D or the 2-bond ( 2 J HN ) HSQC experiments, the absolute enrichment of proton resonances obtained from the 1D 1 H experiments and relative enrichment values obtained from a carbon HSQC spectrum were used to compute the absolute enrichment levels (Walker et al 1982 ; Kishore et al 2005 ).…”

“…However, incorporation of site specific isotopically labeled nucleotides into RNA is necessary to overcome the drawbacks of uniform labeling (Johnson et al 2006 ; Johnson and Hoogstraten 2008 ; Schultheisz et al 2008 ; Dayie and Thakur 2010 ; Thakur et al 2010a , b ; Thakur and Dayie 2011 ) as we and others have demonstrated using RNAs labeled with uniform and selective NTPs (Johnson et al 2006 ; Johnson and Hoogstraten 2008 ; Schultheisz et al 2008 ; Dayie and Thakur 2010 ; Thakur et al 2010a , b ; Thakur and Dayie 2011 ). Previous work had focused on site specific labeling using the gluconeogenic carbon sources of glycerol, glycerol and formate, pyruvate, or acetate using either wild type or two mutant E. coli strains (Hoffman and Holland 1995 ; Johnson et al 2006 ; Johnson and Hoogstraten 2008 ; Dayie and Thakur 2010 ; Thakur et al 2010a , b ; Thakur and Dayie 2011 ), and glucose using wild type E. coli (Kishore et al 2005 ). However because E. coli grows readily on glucose with very good yield compared to the other gluconeogenic carbon precursors (Thakur et al 2010a ), it is attractive to explore glucose as an alternative carbon source.…”

Metabolic labeling: Taking advantage of bacterial pathways to prepare spectroscopically useful isotope patterns in proteins and nucleic acids

“…Statistical labeling has also found use in a number of other contexts, including spectral simplification in solid-state NMR (15) and as an aid to residue type identification to facilitate backbone-directed resonance assignment (16). In a preliminary application to nucleic acids, Kishore et al (17) have used a mixture of C-1 and C-2 labeled glucose as a feedstock for E. coli and found some useful labeling patterns in the resulting ribonucleosides, including a high level of enrichment at ribose C1 0 . Since these authors were primarily attempting to achieve nearrandom isotopic distribution, however, details of the isotopomers produced were not reported.…”