1H and 15N magnetic resonance assignments, secondary structure, and tertiary fold of Escherichia coli DnaJ(1-78).
We report the 1H and 15N 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 15N-labeled DnaJ(1-78), which is a stable, folded fragment. Standard 15N-edited NMR techniques afforded complete assignment of the backbone amide 1H and 15N pairs and partial assignment of the side-chain 1H and 15N atoms. The secondary structure of DnaJ(1-78) was determined from NOE connectivities obtained from 3D 15N-separated and 2D homonuclear NOESY spectra as well as 3JHNH alpha coupling constants obtained from a DQF-COSY spectrum and a 15N-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 alpha-helices and no beta-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.
Hill
R B
RB
Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA.
Flanagan
J M
JM
Prestegard
J H
JH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008961:Models, Structural; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006859:Hydrogen