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Ultra fast MAS solid-state NMR studies of protein-protein interactions in the bacterial replisome

Journal Article


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Abstract


  • Replication of genomic DNA in bacteria involves multiple stable

    and transient interactions among protein subunits constituting

    the replisome. Here we show how ultra-fast magic angle spinning

    (MAS) solid-state NMR (ssNMR) and 1H-detection on either

    deutereted or fully protonated samples, can give new information

    about protein-protein interactions in the E. coli replisome. We

    focused on two fundamental interactions: the first one between

    the C-terminal domain of the single-stranded (ss) DNA binding

    protein and its own DNA-binding site, and the second one

    between the DNA polymerase III subunits a and s. SSB (a tetramer

    of 79 kDa) has a structurally well-defined ssDNA binding

    domain (OB-domain) and an intrinsically disordered C-terminal

    (Ct) domain. Its extreme Ct acidic motif is known to mediate the

    binding of SSB to different DNA processing enzymes and scaffold

    proteins. ssNMR provides, for the first time, residue-specific

    evidence for interaction, in multiple heterogeneous conformations,

    of SSB-Ct with its own ssDNA-binding site. In particular

    we compared spectra from the native protein and from a deletion

    mutant lacking the extreme Ct. This interaction acts as a switch

    that directs recruitment of SSB-binding proteins specifically to

    SSB only when it is bound to ssDNA. Moreover we investigated

    the interaction between the C-terminal domains of a (aCTS

    22.5 kDa) and s (sC16 16 kDa). The structure of the complex is

    so far unknown. We used cell free protein synthesis to produce

    aCTS in the presence of sC16. We analyzed two different samples

    in which each the interacting partners is separately 2H,13C,15N

    labelled. The complete resonance assignment of the proteins in

    the complex opens the way to mapping the protein-protein contacts

    and to the determination of the overall structure

Authors


  •   Felletti, M (external author)
  •   Jehle, S (external author)
  •   Wang, Yao (external author)
  •   Ozawa, Kiyoshi (external author)
  •   Jergic, Slobodan
  •   Otting, Gottfried (external author)
  •   Emsley, Lyndon (external author)
  •   Lesage, A S. (external author)
  •   Dixon, Nicholas E.
  •   Pintacuda, Guido (external author)

Publication Date


  • 2012

Citation


  • Felletti, M., Jehle, S., Wang, Y., Ozawa, K., Jergic, S., Otting, G., Emsley, L., Lesage, A., Dixon, N. & Pintacuda, G. (2012). Ultra fast MAS solid-state NMR studies of protein-protein interactions in the bacterial replisome. The Febs Journal, 279 (1), 472-472.

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=8129&context=engpapers

Ro Metadata Url


  • http://ro.uow.edu.au/engpapers/5198

Number Of Pages


  • 0

Start Page


  • 472

End Page


  • 472

Volume


  • 279

Issue


  • 1

Place Of Publication


  • http://onlinelibrary.wiley.com.ezproxy.uow.edu.au/doi/10.1111/j.1742-4658.2010.08705.x/pdf

Abstract


  • Replication of genomic DNA in bacteria involves multiple stable

    and transient interactions among protein subunits constituting

    the replisome. Here we show how ultra-fast magic angle spinning

    (MAS) solid-state NMR (ssNMR) and 1H-detection on either

    deutereted or fully protonated samples, can give new information

    about protein-protein interactions in the E. coli replisome. We

    focused on two fundamental interactions: the first one between

    the C-terminal domain of the single-stranded (ss) DNA binding

    protein and its own DNA-binding site, and the second one

    between the DNA polymerase III subunits a and s. SSB (a tetramer

    of 79 kDa) has a structurally well-defined ssDNA binding

    domain (OB-domain) and an intrinsically disordered C-terminal

    (Ct) domain. Its extreme Ct acidic motif is known to mediate the

    binding of SSB to different DNA processing enzymes and scaffold

    proteins. ssNMR provides, for the first time, residue-specific

    evidence for interaction, in multiple heterogeneous conformations,

    of SSB-Ct with its own ssDNA-binding site. In particular

    we compared spectra from the native protein and from a deletion

    mutant lacking the extreme Ct. This interaction acts as a switch

    that directs recruitment of SSB-binding proteins specifically to

    SSB only when it is bound to ssDNA. Moreover we investigated

    the interaction between the C-terminal domains of a (aCTS

    22.5 kDa) and s (sC16 16 kDa). The structure of the complex is

    so far unknown. We used cell free protein synthesis to produce

    aCTS in the presence of sC16. We analyzed two different samples

    in which each the interacting partners is separately 2H,13C,15N

    labelled. The complete resonance assignment of the proteins in

    the complex opens the way to mapping the protein-protein contacts

    and to the determination of the overall structure

Authors


  •   Felletti, M (external author)
  •   Jehle, S (external author)
  •   Wang, Yao (external author)
  •   Ozawa, Kiyoshi (external author)
  •   Jergic, Slobodan
  •   Otting, Gottfried (external author)
  •   Emsley, Lyndon (external author)
  •   Lesage, A S. (external author)
  •   Dixon, Nicholas E.
  •   Pintacuda, Guido (external author)

Publication Date


  • 2012

Citation


  • Felletti, M., Jehle, S., Wang, Y., Ozawa, K., Jergic, S., Otting, G., Emsley, L., Lesage, A., Dixon, N. & Pintacuda, G. (2012). Ultra fast MAS solid-state NMR studies of protein-protein interactions in the bacterial replisome. The Febs Journal, 279 (1), 472-472.

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=8129&context=engpapers

Ro Metadata Url


  • http://ro.uow.edu.au/engpapers/5198

Number Of Pages


  • 0

Start Page


  • 472

End Page


  • 472

Volume


  • 279

Issue


  • 1

Place Of Publication


  • http://onlinelibrary.wiley.com.ezproxy.uow.edu.au/doi/10.1111/j.1742-4658.2010.08705.x/pdf