Four-dimensional heteronuclear correlation experiments for chemical shift assignment of solid proteins |
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Authors: | W Trent Franks Kathryn D Kloepper Benjamin J Wylie Chad M Rienstra |
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Institution: | (1) Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA;(2) Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA;(3) Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA |
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Abstract: | 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 CAi], CAi] to Ni],
and Ni] 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. |
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Keywords: | Solid-state NMR Multidimensional NMR Protein structure Magic-angle spinning Chemical shift assignments |
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