1 H and 15 N Magnetic Resonance Assignments, Secondary Structure, and Tertiary Fold of Escherichia coli DnaJ(1-78)

R. Blake Hill, James H. Prestegard, John Flanagan

Research output: Contribution to journalArticle

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Abstract

We report the 1 H and 15 N chemical shift assignments along with an NMR-derived preliminary structure for DnaJ(1-78), a highly conserved N-terminal domain of DnaJ, the Escherichia coli Hsp40 homolog. This 9 kDa domain is believed to cooperate with DnaK, the E. coli Hsp70 homolog, in regulating a variety of cellular functions. Heteronuclear 3D NMR experiments were carried out on a uniformly 15 N-labeled DnaJ(1-78), which is a stable, folded fragment. Standard 15 N-edited NMR techniques afforded complete assignment of the backbone amide 1 H and 15 N pairs and partial assignment of the side-chain and 15 N atoms. The secondary structure of DnaJ(1-78) was determined from NOE connectivities obtained from 3D 15 N-separated and 2D homonuclear NOESY spectra as well as 3 JHNHCX coupling constants obtained from a DQF-COSY spectrum and a 15 N-edited HNHA experiment. The stability of secondary structural elements was assessed by monitoring amide exchange rates, and a model for the three-dimensional fold of these elements was derived from a set of long-range contacts extracted from homonuclear 2D NOESY experiments. The analysis indicates that DnaJ(1-78) is comprised of four a-helices and no β-sheet with a short unstructured loop between antiparallel helices II and III. The shorter N-terminal and C-terminal helices make contacts with helices II and III at points well removed from the central loop. A discussion of how this preliminary structural model may explain mutation data from other laboratories is presented.

Original languageEnglish (US)
Pages (from-to)5587-5596
Number of pages10
JournalBiochemistry
Volume34
Issue number16
DOIs
StatePublished - Apr 1 1995

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Magnetic resonance
Amides
Escherichia coli
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance
Biomolecular Nuclear Magnetic Resonance
Structural Models
Experiments
Chemical shift
Mutation
Atoms
Monitoring

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "1 H and 15 N Magnetic Resonance Assignments, Secondary Structure, and Tertiary Fold of Escherichia coli DnaJ(1-78)",
abstract = "We report the 1 H and 15 N chemical shift assignments along with an NMR-derived preliminary structure for DnaJ(1-78), a highly conserved N-terminal domain of DnaJ, the Escherichia coli Hsp40 homolog. This 9 kDa domain is believed to cooperate with DnaK, the E. coli Hsp70 homolog, in regulating a variety of cellular functions. Heteronuclear 3D NMR experiments were carried out on a uniformly 15 N-labeled DnaJ(1-78), which is a stable, folded fragment. Standard 15 N-edited NMR techniques afforded complete assignment of the backbone amide 1 H and 15 N pairs and partial assignment of the side-chain and 15 N atoms. The secondary structure of DnaJ(1-78) was determined from NOE connectivities obtained from 3D 15 N-separated and 2D homonuclear NOESY spectra as well as 3 JHNHCX coupling constants obtained from a DQF-COSY spectrum and a 15 N-edited HNHA experiment. The stability of secondary structural elements was assessed by monitoring amide exchange rates, and a model for the three-dimensional fold of these elements was derived from a set of long-range contacts extracted from homonuclear 2D NOESY experiments. The analysis indicates that DnaJ(1-78) is comprised of four a-helices and no β-sheet with a short unstructured loop between antiparallel helices II and III. The shorter N-terminal and C-terminal helices make contacts with helices II and III at points well removed from the central loop. A discussion of how this preliminary structural model may explain mutation data from other laboratories is presented.",
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1 H and 15 N Magnetic Resonance Assignments, Secondary Structure, and Tertiary Fold of Escherichia coli DnaJ(1-78) . / Hill, R. Blake; Prestegard, James H.; Flanagan, John.

In: Biochemistry, Vol. 34, No. 16, 01.04.1995, p. 5587-5596.

Research output: Contribution to journalArticle

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T1 - 1 H and 15 N Magnetic Resonance Assignments, Secondary Structure, and Tertiary Fold of Escherichia coli DnaJ(1-78)

AU - Hill, R. Blake

AU - Prestegard, James H.

AU - Flanagan, John

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N2 - We report the 1 H and 15 N chemical shift assignments along with an NMR-derived preliminary structure for DnaJ(1-78), a highly conserved N-terminal domain of DnaJ, the Escherichia coli Hsp40 homolog. This 9 kDa domain is believed to cooperate with DnaK, the E. coli Hsp70 homolog, in regulating a variety of cellular functions. Heteronuclear 3D NMR experiments were carried out on a uniformly 15 N-labeled DnaJ(1-78), which is a stable, folded fragment. Standard 15 N-edited NMR techniques afforded complete assignment of the backbone amide 1 H and 15 N pairs and partial assignment of the side-chain and 15 N atoms. The secondary structure of DnaJ(1-78) was determined from NOE connectivities obtained from 3D 15 N-separated and 2D homonuclear NOESY spectra as well as 3 JHNHCX coupling constants obtained from a DQF-COSY spectrum and a 15 N-edited HNHA experiment. The stability of secondary structural elements was assessed by monitoring amide exchange rates, and a model for the three-dimensional fold of these elements was derived from a set of long-range contacts extracted from homonuclear 2D NOESY experiments. The analysis indicates that DnaJ(1-78) is comprised of four a-helices and no β-sheet with a short unstructured loop between antiparallel helices II and III. The shorter N-terminal and C-terminal helices make contacts with helices II and III at points well removed from the central loop. A discussion of how this preliminary structural model may explain mutation data from other laboratories is presented.

AB - We report the 1 H and 15 N chemical shift assignments along with an NMR-derived preliminary structure for DnaJ(1-78), a highly conserved N-terminal domain of DnaJ, the Escherichia coli Hsp40 homolog. This 9 kDa domain is believed to cooperate with DnaK, the E. coli Hsp70 homolog, in regulating a variety of cellular functions. Heteronuclear 3D NMR experiments were carried out on a uniformly 15 N-labeled DnaJ(1-78), which is a stable, folded fragment. Standard 15 N-edited NMR techniques afforded complete assignment of the backbone amide 1 H and 15 N pairs and partial assignment of the side-chain and 15 N atoms. The secondary structure of DnaJ(1-78) was determined from NOE connectivities obtained from 3D 15 N-separated and 2D homonuclear NOESY spectra as well as 3 JHNHCX coupling constants obtained from a DQF-COSY spectrum and a 15 N-edited HNHA experiment. The stability of secondary structural elements was assessed by monitoring amide exchange rates, and a model for the three-dimensional fold of these elements was derived from a set of long-range contacts extracted from homonuclear 2D NOESY experiments. The analysis indicates that DnaJ(1-78) is comprised of four a-helices and no β-sheet with a short unstructured loop between antiparallel helices II and III. The shorter N-terminal and C-terminal helices make contacts with helices II and III at points well removed from the central loop. A discussion of how this preliminary structural model may explain mutation data from other laboratories is presented.

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