Utility of methyl side chain probes for solution NMR studies of large proteins

Site-Specific Introduction of Alanines for the Nuclear Magnetic Resonance Investigation of Low-Complexity Regions and Large Biomolecular Assemblies

“…The structural and dynamic investigation of these macromolecular machines has been accomplished by using methyl-NMR. − The fast rotation of the methyl group and the presence of three equivalent hydrogen atoms make methyl-NMR experiments especially suited to study large biomolecular assemblies. In order to obtain appropriate samples for these experiments, one or a few methyl-containing amino acids (leucine, isoleucine, valine, alanine, threonine, and methionine) are incorporated in their 13 CH 3 -labeled form, while the rest of the protein is deuterated. − This labeling scheme can be achieved by supplying the bacterial culture with the appropriate isotopologues or with molecules that are precursors in the enzymatic chain of the amino acid synthesis. − However, in methyl-labeled samples, traditional sequential assignment strategies are not possible. For these large machines, mutagenesis-based , or “divide-and-conquer” , approaches are normally applied.…”

“…In order to validate our strategy for alanine-rich LCRs, we applied the alanine SSIL strategy to investigate the two poly-A tracts present at the C-terminal domain of Phox2B (UniProt ID Q99453, residues 136−314). This 179-residue long fragment is a LCR highly enriched in alanines (45), glycines (33), prolines (19), and serines (17). Despite the fact that the resulting 15 N-HSQC spectrum of this fragment N-terminally fused to sfGFP exhibited a large region with overlapped frequencies, several resolved peaks (98) could be identified (Figure S4).…”

Site-Specific Introduction of Alanines for the NMR Investigation of Low-Complexity Regions and Large Biomolecular Assemblies

“…The fast rotation of the methyl group and the presence of three equivalent hydrogen atoms makes methyl-NMR experiments especially suited to study large biomolecular assemblies. In order to obtain appropriate samples for these experiments, one or a few methyl-containing amino acids (leucine, isoleucine, valine, alanine, threonine and methionine) are incorporated in their 13 CH 3 -labeled form, while the rest of the protein is deuterated [15][16][17] . This labeling scheme can be achieved by supplying the bacterial culture with the appropriate isotopologues or with molecules that are precursors in the enzymatic chain of the amino acid synthesis [18][19][20][21] .…”