1H, 15N, 13C, and 13CO Assignments of Human Interleukin-4 Using Three-Dimensional Double- and Triple-Resonance Heteronuclear Magnetic Resonance Spectroscopy

Robert Powers, Daniel S. Garrett, G. Marius Clore, Carl J. March, Eric A. Frieden, Angela M. Gronenbom

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of >95% for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)N(i-1), NH(i-1) correlations are observed in the HNCO experiment, the CαH(i), Cα(i)-N(i+l) correlations in the HCA(CO)N experiment, the Cα(i)-N(i+1), NH(i+1) correlations in the HNCA and HN(CO)CA experiments, the Cα(i)-N(i+1), NH(i+1) correlations in the H(CA)NH and HN(CO)HB experiments, and the Cβ(i)-N(i+1), NH(i+1) correlations in the HN(CO)HB experiments. The backbone intraresidue Cα(i)-15N(i), NH(i)correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the Cβ(i)-15N(i), NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the 13Cα(i)-15N(i), NH(i) correlations by the HNCA experiment, and the CαH(i)-13Cα(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution.

Original languageEnglish (US)
Pages (from-to)4334-4346
Number of pages13
JournalBiochemistry
Volume31
Issue number17
DOIs
StatePublished - Jan 1 1992

Fingerprint

Magnetic resonance spectroscopy
Carbon Monoxide
Interleukin-4
Magnetic Resonance Spectroscopy
Experiments
Biomolecular Nuclear Magnetic Resonance
Isotopes
Spectrum Analysis
Proteins
Yeasts
Chemical shift
Molecular mass
Yeast

ASJC Scopus subject areas

  • Biochemistry

Cite this

1H, 15N, 13C, and 13CO Assignments of Human Interleukin-4 Using Three-Dimensional Double- and Triple-Resonance Heteronuclear Magnetic Resonance Spectroscopy. / Powers, Robert; Garrett, Daniel S.; Clore, G. Marius; March, Carl J.; Frieden, Eric A.; Gronenbom, Angela M.

In: Biochemistry, Vol. 31, No. 17, 01.01.1992, p. 4334-4346.

Research output: Contribution to journalArticle

Powers, Robert ; Garrett, Daniel S. ; Clore, G. Marius ; March, Carl J. ; Frieden, Eric A. ; Gronenbom, Angela M. / 1H, 15N, 13C, and 13CO Assignments of Human Interleukin-4 Using Three-Dimensional Double- and Triple-Resonance Heteronuclear Magnetic Resonance Spectroscopy. In: Biochemistry. 1992 ; Vol. 31, No. 17. pp. 4334-4346.
@article{a99f4207f8a24bad8007df2cdcc4ef73,
title = "1H, 15N, 13C, and 13CO Assignments of Human Interleukin-4 Using Three-Dimensional Double- and Triple-Resonance Heteronuclear Magnetic Resonance Spectroscopy",
abstract = "The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of >95{\%} for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)N(i-1), NH(i-1) correlations are observed in the HNCO experiment, the CαH(i), Cα(i)-N(i+l) correlations in the HCA(CO)N experiment, the Cα(i)-N(i+1), NH(i+1) correlations in the HNCA and HN(CO)CA experiments, the Cα(i)-N(i+1), NH(i+1) correlations in the H(CA)NH and HN(CO)HB experiments, and the Cβ(i)-N(i+1), NH(i+1) correlations in the HN(CO)HB experiments. The backbone intraresidue Cα(i)-15N(i), NH(i)correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the Cβ(i)-15N(i), NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the 13Cα(i)-15N(i), NH(i) correlations by the HNCA experiment, and the CαH(i)-13Cα(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88{\%}), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution.",
author = "Robert Powers and Garrett, {Daniel S.} and Clore, {G. Marius} and March, {Carl J.} and Frieden, {Eric A.} and Gronenbom, {Angela M.}",
year = "1992",
month = "1",
day = "1",
doi = "10.1021/bi00132a026",
language = "English (US)",
volume = "31",
pages = "4334--4346",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "17",

}

TY - JOUR

T1 - 1H, 15N, 13C, and 13CO Assignments of Human Interleukin-4 Using Three-Dimensional Double- and Triple-Resonance Heteronuclear Magnetic Resonance Spectroscopy

AU - Powers, Robert

AU - Garrett, Daniel S.

AU - Clore, G. Marius

AU - March, Carl J.

AU - Frieden, Eric A.

AU - Gronenbom, Angela M.

PY - 1992/1/1

Y1 - 1992/1/1

N2 - The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of >95% for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)N(i-1), NH(i-1) correlations are observed in the HNCO experiment, the CαH(i), Cα(i)-N(i+l) correlations in the HCA(CO)N experiment, the Cα(i)-N(i+1), NH(i+1) correlations in the HNCA and HN(CO)CA experiments, the Cα(i)-N(i+1), NH(i+1) correlations in the H(CA)NH and HN(CO)HB experiments, and the Cβ(i)-N(i+1), NH(i+1) correlations in the HN(CO)HB experiments. The backbone intraresidue Cα(i)-15N(i), NH(i)correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the Cβ(i)-15N(i), NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the 13Cα(i)-15N(i), NH(i) correlations by the HNCA experiment, and the CαH(i)-13Cα(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution.

AB - The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of >95% for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)N(i-1), NH(i-1) correlations are observed in the HNCO experiment, the CαH(i), Cα(i)-N(i+l) correlations in the HCA(CO)N experiment, the Cα(i)-N(i+1), NH(i+1) correlations in the HNCA and HN(CO)CA experiments, the Cα(i)-N(i+1), NH(i+1) correlations in the H(CA)NH and HN(CO)HB experiments, and the Cβ(i)-N(i+1), NH(i+1) correlations in the HN(CO)HB experiments. The backbone intraresidue Cα(i)-15N(i), NH(i)correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the Cβ(i)-15N(i), NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the 13Cα(i)-15N(i), NH(i) correlations by the HNCA experiment, and the CαH(i)-13Cα(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution.

UR - http://www.scopus.com/inward/record.url?scp=0026611088&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026611088&partnerID=8YFLogxK

U2 - 10.1021/bi00132a026

DO - 10.1021/bi00132a026

M3 - Article

C2 - 1567880

AN - SCOPUS:0026611088

VL - 31

SP - 4334

EP - 4346

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 17

ER -