Comprehensive and cost-effective NMR spectroscopy of methyl groups in large proteins.
An NMR approach is described which yields the methyl resonance assignments of alanine, threonine, valine, leucine, and isoleucine residues in proteins with high sensitivity and excellent resolution. The method relies on protein samples produced by bacterial expression using [(1)H,(13)C]-D-glucose and approximately 100% D(2)O, which is cost-effective and ensures the isotopic enrichment of all possible methyl groups. Magnetization transfer throughout the methyl-containing side chains is possible with this labeling scheme due to the high level of deuteration along the amino acid side chain, coupled with the selection of the favorable CHD(2) methyl isotopomer for detection. In an application to the 34 kDa periplasmic binding protein FepB 164 out of 195 methyl groups (85%) were assigned sequence-specifically and stereospecifically. This percentage increases to 91% when taking into account that not all backbone assignments are available for this system. The remaining unassigned methyl groups belong to six leucine residues, caused by low cross-peak intensities, and four alanine residues due to degeneracy of the (13)C(alpha)/(13)C(beta) frequencies. Our results demonstrate that NMR spectroscopic investigations of protein structure, dynamics, and interactions can be extended to include all methyl-containing amino acids also for larger proteins.
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Renee
R
Otten
Byron
B
Chu
Karla D
KD
Krewulak
Hans J
HJ
Vogel
Frans A A
FA
Mulder
D000409:Alanine / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification; D007532:Isoleucine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000592:standards; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification; D033903:Periplasmic Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012015:Reference Standards; D013912:Threonine / Q000737:chemistry; D014633:Valine / Q000737:chemistry
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D033903:Periplasmic Proteins; C061266:fepB protein, E coli; D007532:Isoleucine; D013912:Threonine; D007930:Leucine; D014633:Valine; D000409:Alanine