Enhanced biosynthetically directed fractional carbon-13 enrichment of proteins for backbone NMR assignments

Routes to carbon-13 enrichment of bacterially expressed proteins include achieving uniform or positionally selective (e.g. ILV-Me, or 13C′, etc.) enrichment. We consider the potential for biosynthetically directed fractional enrichment (e.g. carbon-13 incorporation in the protein less than 100%) for performing routine n-(D)dimensional NMR spectroscopy of proteins. First, we demonstrate an approach to fractional isotope addition where the initial growth media containing natural abundance glucose is replenished at induction with a small amount (e.g. 10%w/wu-13C-glucose) of enriched nutrient. The approach considered here is to add 10% (e.g. 200 mg for a 2 g/L culture) u-13C-glucose at the induction time (OD600 = 0.8), resulting in a protein with enhanced 13C incorporation that gives almost the same NMR signal levels as an exact 20% 13C sample. Second, whereas fractional enrichment is used for obtaining stereospecific methyl assignments, we find that 13C incorporation levels no greater than 20%w/w yield 13C and 13C-13C spin pair incorporation sufficient to conduct typical 3D-bioNMR backbone experiments on moderate instrumentation (600 MHz, RT probe). Typical 3D-bioNMR experiments of a fractionally enriched protein yield expected backbone connectivities, and did not show amino acid biases in this work, with one exception. When adding 10% u-13C glucose to expression media at induction, there is poor preservation of 13Cα-13Cβ spin pairs in the amino acids ILV, leading to the absence of Cβ signals in HNCACB spectra for ILV, a potentially useful editing effect. Enhanced fractional carbon-13 enrichment provides lower-cost routes to high throughput protein NMR studies, and makes modern protein NMR more cost-accessible.