Four-dimensional heteronuclear correlation experiments for chemical shift assignment of solid proteins

Trent W. Franks, Kathryn D. Kloepper, Benjamin J. Wylie, Chad M. Rienstra

Research output: Contribution to journalArticlepeer-review


Chemical shift assignment is the first step in all established protocols for structure determination of uniformly labeled proteins by NMR. The explosive growth in recent years of magic-angle spinning (MAS) solid-state NMR (SSNMR) applications is largely attributable to improved methods for backbone and side-chain chemical shift correlation spectroscopy. However, the techniques developed so far have been applied primarily to proteins in the size range of 5-10 kDa, despite the fact that SSNMR has no inherent molecular weight limits. Rather, the degeneracy inherent to many 2D and 3D SSNMR spectra of larger proteins has prevented complete unambiguous chemical shift assignment. Here we demonstrate the implementation of 4D backbone chemical shift correlation experiments for assignment of solid proteins. The experiments greatly reduce spectral degeneracy at a modest cost in sensitivity, which is accurately described by theory. We consider several possible implementations and investigate the CANCOCX pulse sequence in detail. This experiment involves three cross polarization steps, from H to CA[i], CA[i] to N[i], and N[i] to C′[i-1], followed by a final homonuclear mixing period. With short homonuclear mixing times (<20 ms), backbone correlations are observed with high sensitivity; with longer mixing times (>200 ms), long-range correlations are revealed. For example, a single 4D experiment with 225 ms homonuclear mixing time reveals ∼200 uniquely resolved medium and long-range correlations in the 56-residue protein GB1. In addition to experimental demonstrations in the 56-residue protein GB1, we present a theoretical analysis of anticipated improvements in resolution for much larger proteins and compare these results in detail with the experiments, finding good agreement between experiment and theory under conditions of stable instrumental performance.

Original languageEnglish (US)
Pages (from-to)107-131
Number of pages25
JournalJournal of Biomolecular NMR
Issue number2
StatePublished - Oct 2007


  • Chemical shift assignments
  • Magic-angle spinning
  • Multidimensional NMR
  • Protein structure
  • Solid-state NMR

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy


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