Assignment of congested NMR spectra: carbonyl backbone enrichment via the Entner-Doudoroff pathway.
In NMR spectra of complex proteins, sparse isotope enrichment can be important, in that the removal of many (13)C-(13)C homonuclear J-couplings can narrow the lines and thereby facilitate the process of spectral assignment and structure elucidation. We present a simple scheme for selective yet extensive isotopic enrichment applicable for production of proteins in organisms utilizing the Entner-Doudoroff (ED) metabolic pathway. An enrichment scheme so derived is demonstrated in the context of a magic-angle spinning solid-state NMR (MAS SSNMR) study of Pf1 bacteriophage, the host of which is Pseudomonas aeruginosa, strain K (PAK), an organism that uses the ED pathway for glucose catabolism. The intact and infectious Pf1 phage in this study was produced by infected PAK cells grown on a minimal medium containing 1-(13)C d-glucose ((13)C in position 1) as the sole carbon source, as well as (15)NH(4)Cl as the only nitrogen source. The 37MDa Pf1 phage consists of about 93% major coat protein, 1% minor coat proteins, and 6% single-stranded, circular DNA. As a consequence of this composition and the enrichment scheme, the resonances in the MAS SSNMR spectra of the Pf1 sample were almost exclusively due to carbonyl carbons in the major coat protein. Moreover, 3D heteronuclear NCOCX correlation experiments also show that the amino acids leucine, serine, glycine, and tyrosine were not isotopically enriched in their carbonyl positions (although most other amino acids were), which is as expected based upon considerations of the ED metabolic pathway. 3D NCOCX NMR data and 2D (15)N-(15)N data provided strong verification of many previous assignments of (15)N amide and (13)C carbonyl shifts in this highly congested spectrum; both the semi-selective enrichment patterns and the narrowed linewidths allowed for greater certainty in the assignments as compared with use of uniformly enriched samples alone.
Goldbourt
Amir
A
Department of Chemistry, MC3113, Columbia University, New York, NY 10027, USA.
Day
Loren A
LA
McDermott
Ann E
AE
D000465:Algorithms; D025561:Bacteriophage Pf1 / Q000378:metabolism*; D002247:Carbon Isotopes; D020869:Gene Expression Profiling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000821:virology*; D014764:Viral Proteins / Q000378:metabolism*
D002247:Carbon Isotopes; D014764:Viral Proteins