Solid-state NMR spectroscopy of 10% 13C labeled ubiquitin: spectral simplification and stereospecific assignment of isopropyl groups

Abstract

We describe the simplification of 13C-13C correlation spectra obtained from a microcrystalline protein sample expressed on a growth medium of 10% fully 13C labeled glucose diluted in 90% natural abundance glucose as compared to a fully labeled sample. Such a labeling scheme facilitates the backbone and side-chain resonance assignment of Phe, Tyr, His, Asp, Asn, Ile, Lys and Pro and yields an unambiguous stereospecific assignment of the valine Cgamma1, Cgamma2 13C resonances and of Leucine Cdelta2.

BSH-CP based 3D solid-state NMR experiments for protein resonance assignment

“…The last six C-terminal residues cannot be assigned due to dynamic disorder. In previous studies of MPD-crystallized ubiquitin by ssNMR, only a single set of resonances was reported (Igumenova et al 2004;Schubert et al 2006). One of the reasons for this may lie in the slightly different crystallization conditions used.…”

Metabolic labeling: Taking advantage of bacterial pathways to prepare spectroscopically useful isotope patterns in proteins and nucleic acids

“…In these cases, a bacterial culture is grown up in the absence of protein overexpression, cellular RNA or DNA is isolated and hydrolyzed to monomeric units, the resulting monomers are rephosphorylated to nucleoside triphosphates (NTPs), and the ribo-or deoxyribo-NTPs are used as feedstocks for in vitro enzymatic synthesis of the sequence of interest (8,9). (For purposes of uniform labeling, 15 N-and 13 C, 15 N-NTPs have recently become commercially available, although at considerable expense). Although this procedure is more involved than that for proteins, it is in some senses more general for the schemes described in this review, since transformation and overexpression in a mutant strain is unnecessary.…”

“…Statistical labeling has also found use in a number of other contexts, including spectral simplification in solid-state NMR (15) and as an aid to residue type identification to facilitate backbone-directed resonance assignment (16). In a preliminary application to nucleic acids, Kishore et al (17) have used a mixture of C-1 and C-2 labeled glucose as a feedstock for E. coli and found some useful labeling patterns in the resulting ribonucleosides, including a high level of enrichment at ribose C1 0 .…”

“…The NOE constraints obtained using the backbone NH and side-chain methyl protons only were sufficient to derive a globally folded structure of MBP (40). In short, the reintroduction of protons in the ILV scheme, combined with otherwise uniform 13 C, 15 N, and 2 H labeling and spectroscopy in H 2 O solvent, represented a significant advance in protein NMR spectroscopy by demonstrating resonance assignments and low-resolution structure determination for a protein significantly larger than those analyzed previously.…”

“…In this protocol, every atom of the naturally-occurring isotope of a given element or elements is replaced by an isotope chosen for spectro-scopic purposes- 13 C or 15 N to provide spin-1 2 NMR probes, for example, or 2 H for more specialized purposes described later. Increasingly, however, sophisticated magnetic-resonance methods require the labeling of specific subsets of atoms within an amino acid or nucleotide.…”