Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity

Abstract

A selective isotope labeling scheme based on the utilization of [2-13C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state 13Cα chemical shifts using Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-13C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state 13Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s−1, despite the small fraction of 13Cα–13Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using 13Cα spin probes.<p>At the time for thesis presentation publication was in status: Manuscript</p><p>At the time for thesis presentation name of publication was: Fractional enrichment using [2-¹³C]-glycerol as the carbon source facilitates measurements of excited state ¹³Cα chemical shifts with improved sensitivity</p

Late metabolic precursors for selective aromatic residue labeling

“…Early metabolic precursors (biosynthetic intermediates upstream of the shikimate- or the pentose phosphate pathway) have been applied to achieve a certain isotope distribution in the corresponding target residues. Isotopologues of d -glucose (Teilum et al 2006 ; Lundström et al 2007 , 2009a , b ; Weininger et al 2012a , 2013 ), glycerol (Ahlner et al 2015 ; LeMaster and Kushlan 1996 ; Takeuchi et al 2008 ), d -erythrose (Kasinath et al 2013 , 2015 ; Weininger 2017b ), d -ribose (Weininger 2017a ), acetate (Wand et al 1995 ) and pyruvate (Guo et al 2009 ; Lee et al 1997 ; Lundström et al 2008 ; Milbradt et al 2015 ; Robson et al 2018 ) have been particularly used to reduce unwanted 1 J CC coupling in NMR-based analysis of protein dynamics. These methods have been frequently applied by the biomolecular NMR community, since the corresponding precursors are commercially available and additional synthetic organic chemistry is not needed.…”

Anthranilic acid, the new player in the ensemble of aromatic residue labeling precursor compounds

“…Scheme 1 summarizes the main metabolites, which have been used to overexpress proteins containing labeled phenylalanine, tyrosine or tryptophan residues. Certain 13 C-patterns of D-glucose (Teilum et al 2006 ; Lundström et al 2007 ), acetate (Wand et al 1995 ), erythrose (Kasinath et al 2013 ; Weininger 2017 ), pyruvate (Guo et al 2009 ; Milbradt et al 2015 ), or glycerol (LeMaster and Kushlan 1996 ; Ahlner et al 2015 ) have been applied as carbon sources to avoid direct 13 C– 13 C coupling in the target residues. These compounds are commercially available and additional organic synthesis is usually not required.…”

Site-selective 13C labeling of histidine and tryptophan using ribose

“…In the most general way site-selective 13 C labeling is achieved using glucose (Lundstrom et al 2007 ; Teilum et al 2006 ), glycerol (Ahlner et al 2015 ), or pyruvate (Milbradt et al 2015 ). These labeling schemes with precursors at the beginning of the biological pathways in bacteria, label many positions in all amino acids.…”