Biosynthetic 13C labeling of aromatic side chains in proteins for NMR relaxation measurements.
Site-specific 13C labeling offers a desirable means of eliminating unwanted relaxation pathways and coherent magnetization transfer in NMR relaxation experiments. Here we use [1-13C]-glucose as the sole carbon source in the growth media for protein overexpression in Escherichia coli. The approach results in specific incorporation of 13C at isolated positions in the side chains of aromatic amino acids, which greatly simplifies the measurements and interpretation of 13C relaxation rates in these spin systems. The method is well suited for characterization of chemical exchange by CPMG or spin-lock relaxation methods. We validated the method by acquiring 13C rotating-frame relaxation dispersion data on the E140Q mutant of the C-terminal domain of calmodulin, which reveal conformational exchange dynamics with a time constant of 71 mus for Y138.
Department of Biophysical Chemistry, Lund University, Sweden. kaare.tellum@bpc.lu.se
Kaare
K
Teilum
Ulrika
U
Brath
Patrik
P
Lundström
Mikael
M
Akke
D000214:Acyl Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D024322:Amino Acids, Aromatic / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes; D002417:Cattle; D004926:Escherichia coli / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary
D000214:Acyl Coenzyme A; D024322:Amino Acids, Aromatic; D002147:Calmodulin; D002247:Carbon Isotopes; D010649:Phenylalanine; D005947:Glucose