Assignment of congested NMR spectra: Carbonyl backbone enrichment via the Entner–Doudoroff pathway Amir Goldbourt a , Loren A. Day b , Ann E. McDermott a, * a Department of Chemistry MC3113, Columbia University, New York, NY 10027, USA b Public Health Research Institute, 225 Warren Street, Newark, NJ 07103, USA Received 22 April 2007; revised 10 July 2007 Available online 17 August 2007 Abstract 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) met- abolic 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 37 MDa 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 pre- vious 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. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Pf1 filamentous phage; Entner–Doudoroff pathway; Solid-state NMR; Magic-angle spinning; Resonance assignment; Isotope enrichment 1. Introduction The assignment of NMR resonances in biological mac- romolecules is typically a prerequisite for obtaining site- specific information. In recent years, a growing number of assignments have been reported for globular and mem- brane proteins studied by magic-angle spinning (MAS 1 ) solid-state NMR, yielding several structures [1–5]. One of the key factors for successful assignment is the spectral res- olution. For proteins of large molecular weight or for pro- teins containing a dominant secondary structure element (i.e., all helical or all sheet), spectral congestion and peak overlap can hinder successful assignment. Intrinsic mem- brane proteins, which are both large and mostly helical, provide examples of this problem. Although successful assignments have been reported in some cases [6,7], and partial site-specific assignments and amino acids type assignments of relatively narrow line widths for isolated peaks (0.5 ppm) have also been reported [8–10], spectral overlaps hampered large-scale site-specific assignments. Several methods have been used to overcome the problem of poorly resolved peaks. Larger signal dispersion has 1090-7807/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jmr.2007.07.011 * Corresponding author. Fax: +1 212 932 1289. E-mail address: aem5@columbia.edu (A.E. McDermott). 1 Abbreviations used: ATCC, the global bioresource center (http:// www.atcc.org/Home.cfm); CSA, chemical shift anisotropy; DARR, dipo- lar assisted rotational resonance; ED, Entner–Doudoroff; EMP, Embden– Meyerhoff–Parnas; MAS SSNMR, magic-angle spinning solid-state NMR; NCOCX, 3D heteronuclear NMR experiment for sequential assignment; PAK, Pseudomonas aeruginosa strain K. www.elsevier.com/locate/jmr Available online at www.sciencedirect.com Journal of Magnetic Resonance 189 (2007) 157–165