Stereospecific assignment of the methyl ¹H NMR lines of valine and leucine in polypeptides by nonrandom ¹³C labelling

Abstract

An alternative method for the stereospecific assignment of the methyl groups of valine and leucine in the nuclear magnetic resonance (NMR) spectra of peptides and proteins is proposed, and its practical application is demonstrated with the assignment of all valine and leucine methyl groups in cyclosporin A. The method is based on the use of a mixture of fully i3C-labelled and unlabelled glucose as the sole carbon source for the biosynthetic production of the polypeptide studied, knowledge of the independently established stereoselectivity of the reaction pathways of valine and leucine biosynthesis, and analysis of the distribution of W labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments.

Solution structure of villin 14T, a domain conserved among actin‐severing proteins

“…Dihedral angle restraints for the backbone angle 4 were based on measurements of the vicinal coupling constant 3JH,H,, Restraints on the side chain torsion angle xl and stereospecific assignments for P-methylene protons were derived from measurements of the vicinal coupling constants 3 J H a H B and JNHa. Stereospecific assignments for the methyl groups of valine and leucine were obtained from a I3C heteronuclear single quantum coherence (13C-HSQC) spectrum recorded on a 10% labeled sample (Neri et al, 1989;Senn et al, 1989;Szyperski et al, 1992). The restraints described above were supplemented with 86 general distance restraints representing 43 backbone hydrogen bonds assigned on the basis of hydrogen exchange experiments.…”

“…Stereospecific assignments for methyl groups of valine and leucine were obtained from spectra acquired on the 10% %labeled sample (Neri et al, 1989;Senn et al, 1989;Szyperski et al, 1992).…”

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

“…The 'out-and-back' 3D HMCM(CG)CBCA experiment 21 was acquired on a 0.4-mM methyl-protonated {Ile(d1 13 15 N] sample of PmrA for assigning methyl groups. Stereospecific assignment of methyl groups of Leu and Val residues was as described 41 . Briefly, we used the mixture of 10% 13 C-glucose and 90% unlabelled glucose as the carbon source for the expression of the PmrA sample and analysis of the distribution of 13 C labels of the methyl groups in Leu and Val residues on a 2D 1 H, 13 C HSQC spectrum.…”

Backbone-independent nucleic acid binding by splicing factor SUP-12 reveals key aspects of molecular recognition

“…Glutamine and asparagine sidechain amides were assigned using spectra from a 3D HNCO, 3D HNCACB, 3D CBCA(CO)NH and confirmed using a 3D 15 N-NOESY (120 ms mixing time). Stereospecific assignments for valine and leucine methyl groups were achieved using a 2D constant-time 13 C-HSQC and a 10% uniform-13 C labelling scheme 33 . DNA assignments were based on double-filtered 2D 1 H, 1 H-TOCSY (40 and 120 ms mixing times) and 1 H, 1 H-NOESY (120 ms mixing time) spectra on a sample of 13 C, 15 N-labelled SUP-12 in complex with unlabelled ligand, as well as 2D 13 C-HMQC and 2D 15 N-HSQC spectra with site-specific incorporation of 13 C, 15 N-labelled nucleotides by using chemically synthesized DNA.…”