Assignment of methyl NMR resonances of a 52 kDa protein with residue-specific 4D correlation maps
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Methyl groups have become key probes for structural and functional studies by nuclear magnetic resonance. However, their NMR signals cluster in a small spectral region and assigning their resonances can be a tedious process. Here, we present a method that facilitates assignment of methyl resonances from assigned amide groups. Calculating the covariance between sensitive methyl and amide 3D spectra, each providing correlations to Cα and Cβ separately, produces 4D correlation maps directly correlating methyl groups to amide groups. Optimal correlation maps are obtained by extracting residue-specific regions, applying derivative to the dimensions subject to covariance, and multiplying 4D maps stemming from different 3D spectra. The latter procedure rescues weak signals that may be missed in traditional assignment procedures. Using these covariance correlation maps, nearly all assigned isoleucine, leucine, and valine amide resonances of a 52 kDa nonribosomal peptide synthetase cyclization domain were paired with their corresponding methyl groups.
KeywordsNMR resonance assignment Large proteins Methyl groups Nonribosomal peptide synthetases
We thank Dr Lewis Kay for providing the pulse sequences of HMCMCGCB and HMCM(CG)CBCA, and Brad Harden, Scott Nichols and Andy Goodrich for carefully reading the manuscript. This research was supported by the National Institutes of Health (Grant GM 104257).
- Grzesiek S, Bax A (1993) Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins. J Biomol NMR 3:185–204Google Scholar
- Matlab reference guide (1992). Natick, MAGoogle Scholar
- Mootz HD, Schwarzer D, Marahiel MA (2002) Ways of assembling complex natural products on modular nonribosomal peptide synthetases. Chembiochem 3:490–504. doi: 10.1002/1439-7633(20020603)3:6<490:AID-CBIC490>3.0.CO;2-N CrossRefGoogle Scholar