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Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins

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Abstract

The solution NMR resonance assignment of the protein backbone is most commonly carried out using triple resonance experiments that involve 15N and 1HN resonances. The assignment becomes problematic when there is resonance overlap of 15N–1HN cross peaks. For such residues, one cannot unambiguously link the “left” side of the NH root to the “right” side, and the residues associated with such overlapping HN resonances remain often unassigned. Here we present a solution to this problem: a hybrid (4d,3d) reduced-dimensionality HN(CO)CA(CON)CA sequence. In this experiment, the Ca(i) resonance is modulated with the frequency of the Ca(i−1) resonance, which helps in resolving the ambiguity involved in connecting the Ca(i) and Ca(i−1) resonances for overlapping NH roots. The experiment has limited sensitivity, and is only suited for small or unfolded proteins. In a companion experiment, (4d,3d) reduced-dimensionality HNCO(N)CA, the Ca(i) resonance is modulated with the frequency of the CO(i−1) resonance, hence resolving the ambiguity existent in pairing up the Ca(i) and CO(i−1) resonances for overlapping NH roots.

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Acknowledgments

We acknowledge NIH R01HL 102662; ERPZ also acknowledges NIH ARRA GM063027-S2.

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Authors and Affiliations

  1. Department of Biological Chemistry, University of Michigan Medical School, MSRB III, 1150 W. Medical Center Drive, Ann Arbor, MI, 48109-5605, USA

    Ireena Bagai & Stephen W. Ragsdale

  2. Department of Biological Chemistry, University of Michigan Medical School, 4220D MSRB III, 1150 W. Medical Center Drive, Ann Arbor, MI, 48109-5605, USA

    Erik R. P. Zuiderweg

Authors
  1. Ireena Bagai
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  2. Stephen W. Ragsdale
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  3. Erik R. P. Zuiderweg
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Correspondence to Erik R. P. Zuiderweg.

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Bagai, I., Ragsdale, S.W. & Zuiderweg, E.R.P. Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins. J Biomol NMR 49, 69–74 (2011). https://doi.org/10.1007/s10858-010-9465-1

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  • DOI: https://doi.org/10.1007/s10858-010-9465-1

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