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@INPROCEEDINGS{Hung:172107,
      author       = {Hung, Yu-Fu and Schwarten, Melanie and Schünke, Sven and
                      Hoffmann, Silke and Willbold, Dieter and Sklan, ella and
                      König, Bernd},
      title        = {{S}tructure and membrane interaction of the {N}-terminal
                      region of {D}engue virus {NS}4{A} protein},
      reportid     = {FZJ-2014-05650},
      year         = {2014},
      abstract     = {Dengue virus (DENV) infection presents a serious public
                      health threat with more than one third of the world
                      population at risk. DENV is a mosquito-transmitted virus
                      that causes dengue fever, dengue hemorrhagic fever and
                      dengue shock syndrome. There is no vaccine available against
                      DENV and no specific treatment for dengue fever. DENV is
                      believed to replicate its RNA genome in association with
                      modified intracellular membranes. However, the details of
                      the assembly of this replication complex are incompletely
                      understood. We focused on the DENV non-structural protein 4A
                      (NS4A) which has been implicated in the formation of the
                      viral RNA replication complex. Sequence analysis identified
                      conserved regions in the N-terminal 48 amino acids of NS4A
                      that might form amphipathic helices (AH). Mutations (L6E;
                      M10E) designed to reduce the amphipathic character of the
                      predicted AH, abolished viral replication and reduced NS4A
                      oligomerization [1]. However, little is known about the
                      three dimensional structure of NS4A(1-48). We used solution
                      state nuclear magnetic resonance (NMR) and circular
                      dichroism (CD) spectroscopy to study the structure of wild
                      type NS4A(1-48) and of the double mutant NS4A(1-48,
                      L6E;M10E) in the presence and absence of model membranes.
                      The hydrodynamic radius of liposomes and detergent micelles
                      was determined by dynamic light scattering (DLS). Both
                      peptides were recombinantly produced in E.coli and are
                      basically unstructured in aqueous buffer [2]. Addition of
                      liposomes made of POPC or POPC/DOPS mixtures induced
                      formation of α-helical secondary structure in case of the
                      wild type NS4A(1-48) but not for the mutant peptide. The
                      degree of helicity of wt NS4A(1-48) is sensitive to the
                      lipid composition and to the size of the liposomes.
                      Formation of α-helical secondary structure was observed for
                      both wt and mutant NS4A(1-48) upon addition of various
                      membrane mimicking detergent micelles (SDS, DPC, DHPC, DM).
                      The degree of helix formation depends on the type and
                      concentration of the detergent and reaches a maximum at
                      about 100 mM SDS or DPC. Solution state NMR spectroscopy
                      provided a detailed picture of the structure and micelle
                      interaction for both peptides in presence of 100 mM SDS.
                      Backbone resonance assignment followed by analysis of
                      secondary chemical shifts allowed us to identify two
                      α-helical segments in each peptide which cover amino acid
                      residues 5-10 and 15-29 in NS4A(1-48) and residues 4-9 and
                      15-29 in NS4A(1-48, L6E;M10E). Analysis of paramagnetic
                      relaxation enhancement after addition of paramagnetic Mn2+
                      to the SDS micelle-containing buffer allowed us to
                      distinguish buffer exposed from buried amino acid
                      residues.[1] O. Stern et al. (2013) J. Virol. 87:4080-85[2]
                      Y.F. Hung et al. (2014) PLoS One. 9: e86482},
      month         = {Sep},
      date          = {2014-09-14},
      organization  = {Annual Meeting of the German
                       Biophysical Society, Lübeck (Germany),
                       14 Sep 2014 - 17 Sep 2014},
      subtyp        = {After Call},
      cin          = {ICS-6},
      cid          = {I:(DE-Juel1)ICS-6-20110106},
      pnm          = {452 - Structural Biology (POF2-452) / 553 - Physical Basis
                      of Diseases (POF3-553)},
      pid          = {G:(DE-HGF)POF2-452 / G:(DE-HGF)POF3-553},
      typ          = {PUB:(DE-HGF)24},
      url          = {https://juser.fz-juelich.de/record/172107},
}