Triple-resonance NOESY-based experiments with improved spectral resolution

Applications to structural characterization of unfolded, partially folded and folded proteins

Ouwen Zhang, Julie D. Forman-Kay, David R Shortle, Lewis E. Kay

Research output: Contribution to journalArticle

Abstract

NMR-based structural studies of macromolecules focus to a large extent on the establishment of interproton distances within the molecule based on the nuclear Overhauser effect (NOE). Despite the improvements in resolution resulting from multidimensional NMR experiments, the detailed characterization of disordered states of proteins or highly overlapped regions of folded molecules using current NMR methods remains challenging. A suite of triple-resonance NOESY-type pulse schemes is presented which require uniform 15N and 13C labeling and make use of the chemical shift dispersion of backbone 15N and 13C′ (carbonyl) resonances to increase the spectral resolution. In particular, for the case of partially folded and unfolded proteins, the experiments exploit the fact that the dispersion of 15N and 13C′ resonances is comparable to that observed in folded states. Ambiguities that arise in the assignment of NOEs as a result of the severe chemical shift degeneracy in ́H and aliphatic 13C nuclei are resolved, therefore, by recording the chemical shifts of 15N or 13C′ either before or after the NOE mixing period. Applications of these methods to the study of the unfolded state of the N-terminal SH3 domain of drk (drkN SH3) and a partially folded large fragment of staphylococcal nuclease (SNase), Δ131Δ, are presented. In addition, an application to folded SNase in complex with the ligands thymidine 3′,5′-bisphosphate (pdTp) and Ca2+ is illustrated which allows the assignment of NOEs between degenerate Hα protons or protons resonating close to water.

Original languageEnglish (US)
Pages (from-to)181-200
Number of pages20
JournalJournal of Biomolecular NMR
Volume9
Issue number2
StatePublished - 1997

Fingerprint

Micrococcal Nuclease
Spectral resolution
Chemical shift
Protons
Nuclear magnetic resonance
Protein Unfolding
src Homology Domains
Thymidine
Proteins
Molecules
Experiments
Ligands
Macromolecules
Labeling
Water
thymidine 3',5'-diphosphate

Keywords

  • Heteronuclear
  • Multidimensional NMR
  • NOE
  • Unfolded or partially folded proteins

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Spectroscopy

Cite this

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title = "Triple-resonance NOESY-based experiments with improved spectral resolution: Applications to structural characterization of unfolded, partially folded and folded proteins",
abstract = "NMR-based structural studies of macromolecules focus to a large extent on the establishment of interproton distances within the molecule based on the nuclear Overhauser effect (NOE). Despite the improvements in resolution resulting from multidimensional NMR experiments, the detailed characterization of disordered states of proteins or highly overlapped regions of folded molecules using current NMR methods remains challenging. A suite of triple-resonance NOESY-type pulse schemes is presented which require uniform 15N and 13C labeling and make use of the chemical shift dispersion of backbone 15N and 13C′ (carbonyl) resonances to increase the spectral resolution. In particular, for the case of partially folded and unfolded proteins, the experiments exploit the fact that the dispersion of 15N and 13C′ resonances is comparable to that observed in folded states. Ambiguities that arise in the assignment of NOEs as a result of the severe chemical shift degeneracy in ́H and aliphatic 13C nuclei are resolved, therefore, by recording the chemical shifts of 15N or 13C′ either before or after the NOE mixing period. Applications of these methods to the study of the unfolded state of the N-terminal SH3 domain of drk (drkN SH3) and a partially folded large fragment of staphylococcal nuclease (SNase), Δ131Δ, are presented. In addition, an application to folded SNase in complex with the ligands thymidine 3′,5′-bisphosphate (pdTp) and Ca2+ is illustrated which allows the assignment of NOEs between degenerate Hα protons or protons resonating close to water.",
keywords = "Heteronuclear, Multidimensional NMR, NOE, Unfolded or partially folded proteins",
author = "Ouwen Zhang and Forman-Kay, {Julie D.} and Shortle, {David R} and Kay, {Lewis E.}",
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language = "English (US)",
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journal = "Journal of Biomolecular NMR",
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T1 - Triple-resonance NOESY-based experiments with improved spectral resolution

T2 - Applications to structural characterization of unfolded, partially folded and folded proteins

AU - Zhang, Ouwen

AU - Forman-Kay, Julie D.

AU - Shortle, David R

AU - Kay, Lewis E.

PY - 1997

Y1 - 1997

N2 - NMR-based structural studies of macromolecules focus to a large extent on the establishment of interproton distances within the molecule based on the nuclear Overhauser effect (NOE). Despite the improvements in resolution resulting from multidimensional NMR experiments, the detailed characterization of disordered states of proteins or highly overlapped regions of folded molecules using current NMR methods remains challenging. A suite of triple-resonance NOESY-type pulse schemes is presented which require uniform 15N and 13C labeling and make use of the chemical shift dispersion of backbone 15N and 13C′ (carbonyl) resonances to increase the spectral resolution. In particular, for the case of partially folded and unfolded proteins, the experiments exploit the fact that the dispersion of 15N and 13C′ resonances is comparable to that observed in folded states. Ambiguities that arise in the assignment of NOEs as a result of the severe chemical shift degeneracy in ́H and aliphatic 13C nuclei are resolved, therefore, by recording the chemical shifts of 15N or 13C′ either before or after the NOE mixing period. Applications of these methods to the study of the unfolded state of the N-terminal SH3 domain of drk (drkN SH3) and a partially folded large fragment of staphylococcal nuclease (SNase), Δ131Δ, are presented. In addition, an application to folded SNase in complex with the ligands thymidine 3′,5′-bisphosphate (pdTp) and Ca2+ is illustrated which allows the assignment of NOEs between degenerate Hα protons or protons resonating close to water.

AB - NMR-based structural studies of macromolecules focus to a large extent on the establishment of interproton distances within the molecule based on the nuclear Overhauser effect (NOE). Despite the improvements in resolution resulting from multidimensional NMR experiments, the detailed characterization of disordered states of proteins or highly overlapped regions of folded molecules using current NMR methods remains challenging. A suite of triple-resonance NOESY-type pulse schemes is presented which require uniform 15N and 13C labeling and make use of the chemical shift dispersion of backbone 15N and 13C′ (carbonyl) resonances to increase the spectral resolution. In particular, for the case of partially folded and unfolded proteins, the experiments exploit the fact that the dispersion of 15N and 13C′ resonances is comparable to that observed in folded states. Ambiguities that arise in the assignment of NOEs as a result of the severe chemical shift degeneracy in ́H and aliphatic 13C nuclei are resolved, therefore, by recording the chemical shifts of 15N or 13C′ either before or after the NOE mixing period. Applications of these methods to the study of the unfolded state of the N-terminal SH3 domain of drk (drkN SH3) and a partially folded large fragment of staphylococcal nuclease (SNase), Δ131Δ, are presented. In addition, an application to folded SNase in complex with the ligands thymidine 3′,5′-bisphosphate (pdTp) and Ca2+ is illustrated which allows the assignment of NOEs between degenerate Hα protons or protons resonating close to water.

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