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

Abstract

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. 200mg for a 2g/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 (600MHz, 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.