1-13C amino acid selective labeling in a 2H15N background for NMR studies of large proteins.
Isotope labeling by residue type (LBRT) has long been an important tool for resonance assignments at the limit where other approaches, such as triple-resonance experiments or NOESY methods do not succeed in yielding complete assignments. While LBRT has become less important for small proteins it can be the method of last resort for completing assignments of the most challenging protein systems. Here we present an approach where LBRT is achieved by adding protonated (14)N amino acids that are (13)C labeled at the carbonyl position to a medium for uniform deuteration and (15)N labeling. This has three important benefits over conventional (15)N LBRT in a deuterated back ground: (1) selective TROSY-HNCO cross peaks can be observed with high sensitivity for amino-acid pairs connected by the labeling, and the amide proton of the residue following the (13)C labeled amino acid is very sharp since its alpha position is deuterated, (2) the (13)C label at the carbonyl position is less prone to scrambling than the (15)N at the alpha-amino position, and (3) the peaks for the 1-(13)C labeled amino acids can be identified easily from the large intensity reduction in the (1)H-(15)N TROSY-HSQC spectrum for some residues that do not significantly scramble nitrogens, such as alanine and tyrosine. This approach is cost effective and has been successfully applied to proteins larger than 40 kDa.
Takeuchi
Koh
K
Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Ng
Elise
E
Malia
Thomas J
TJ
Wagner
Gerhard
G
D000596:Amino Acids / Q000737:chemistry*; D002247:Carbon Isotopes / Q000737:chemistry; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results
D000596:Amino Acids; D002247:Carbon Isotopes; D011506:Proteins