Resonance assignments and secondary structure analysis of E. coli thioredoxin by magic angle spinning solid-state NMR spectroscopy.
De novo site-specific 13C and 15N backbone and sidechain resonance assignments are presented for uniformly enriched E. coli thioredoxin, established using two-dimensional homo- and heteronuclear solid-state magic angle spinning NMR correlation spectroscopy. Backbone dihedral angles and secondary structure were derived from the statistical analysis of the secondary chemical shifts, and are in good agreement with solution values for the intact full-length thioredoxin, with the exception of a small number of residues located at the termini of the individual secondary structure elements. A large number of cross-peaks observed in the DARR spectra with long mixing times correspond to the pairs of carbon atoms separated by 4-6 angstroms, suggesting that DARR could be efficiently employed for observation of medium- and long-range correlations. The 108 amino acid residue E. coli thioredoxin is the largest uniformly enriched protein assigned to this degree of completeness by solid-state NMR spectroscopy to date. It is anticipated that with a combination of two-dimensional correlation experiments and high magnetic fields, resonance assignments and secondary structure can be generally derived for other noncrystalline proteins.
Marulanda
Dabeiba
D
Department of Chemistry and Biochemistry, Brown Laboratories, University of Delaware, Newark, Delaware 19716, USA.
Tasayco
Maria Luisa
ML
Cataldi
Marcela
M
Arriaran
Vilma
V
Polenova
Tatyana
T
D002247:Carbon Isotopes; D003460:Crystallization; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D011092:Polyethylene Glycols; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013879:Thioredoxins / Q000737:chemistry*
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D011092:Polyethylene Glycols; D013879:Thioredoxins