High-resolution methyl edited GFT NMR experiments for protein resonance assignments and structure determination.
Three-dimensional (3D) structure determination of proteins is benefitted by long-range distance constraints comprising the methyl groups, which constitute the hydrophobic core of proteins. However, in methyl groups (of Ala, Ile, Leu, Met, Thr and Val) there is a significant overlap of ¹³C and ¹H chemical shifts. Such overlap can be resolved using the recently proposed (3,2)D HCCH-COSY, a G-matrix Fourier transform (GFT) NMR based experiment, which facilitates editing of methyl groups into distinct spectral regions by combining their ¹³C chemical shifts with that of the neighboring, directly attached, ¹³C nucleus. Using this principle, we present three GFT experiments: (a) (4,3)D NOESY-HCCH, (b) (4,3)D ¹H-TOCSY-HCCH and (c) (4,3)D ¹³C-TOCSY-HCCH. These experiments provide unique 4D spectral information rapidly with high sensitivity and resolution for side-chain resonance assignments and NOE analysis of methyl groups. This is exemplified by (4,3)D NOESY-HCCH data acquired for 17.9 kDa non-deuterated cytosolic human J-protein co-chaperone, which provided crucial long-range distance constraints for its 3D structure determination.
Jaipuria
Garima
G
NMR Research Centre, Indian Institute of Science, Bangalore, India.
Thakur
Anushikha
A
D'Silva
Patrick
P
Atreya
Hanudatta S
HS
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D003903:Deuterium; D004926:Escherichia coli / Q000737:chemistry / Q000254:growth & development; D005583:Fourier Analysis; D006801:Humans; D007532:Isoleucine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D008745:Methylation; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000737:chemistry*; D014633:Valine / Q000737:chemistry
D002247:Carbon Isotopes; D011506:Proteins; D007532:Isoleucine; D003903:Deuterium; D007930:Leucine; D014633:Valine