title: 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 (13)C', 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/w)u-(13)C-glucose) of enriched nutrient. The approach considered here is to add 10% (e.g. 200mg for a 2g/L culture) u-(13)C-glucose at the induction time (OD600=0.8), resulting in a protein with enhanced (13)C incorporation that gives almost the same NMR signal levels as an exact 20% (13)C sample. Second, whereas fractional enrichment is used for obtaining stereospecific methyl assignments, we find that (13)C incorporation levels no greater than 20%(w/w) yield (13)C and (13)C-(13)C 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-(13)C glucose to expression media at induction, there is poor preservation of (13)Cα-(13)Cβ 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.
authors.affiliation: Bucknell University, Lewisburg, PA 17837, United States.
authors.forename: Broc R
authors.initials: BR
authors.lastname: Wenrich
authors.affiliation: Bucknell University, Lewisburg, PA 17837, United States.
authors.forename: Reilly E
authors.initials: RE
authors.lastname: Sonstrom
authors.affiliation: Bucknell University, Lewisburg, PA 17837, United States.
authors.forename: Riju A
authors.initials: RA
authors.lastname: Gupta
authors.affiliation: Bucknell University, Lewisburg, PA 17837, United States. Electronic address: drovnyak@bucknell.edu.
authors.forename: David
authors.initials: D
authors.lastname: Rovnyak
mesh_terms: D000597:Amino Acids, Branched-Chain / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D025801:Ubiquitin
chemical_list: D000597:Amino Acids, Branched-Chain; D002247:Carbon Isotopes; D005656:Fungal Proteins; D011506:Proteins; D025801:Ubiquitin
keywords: Biosynthetically directed fractional enrichment; Protein NMR; Yeast ubiquitin