Domain Swapping Proceeds via Complete Unfolding: A F-19- and H-1-NMR Study of the Cyanovirin-N Protein

Department of Structural Biology, University of Pittsburgh School of Medicine, 1051 Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, Pennsylvania 15261, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 03/2012; 134(9):4229-35. DOI: 10.1021/ja210118w
Source: PubMed


Domain swapping creates protein oligomers by exchange of structural units between identical monomers. At present, no unifying molecular mechanism of domain swapping has emerged. Here we used the protein Cyanovirin-N (CV-N) and (19)F-NMR to investigate the process of domain swapping. CV-N is an HIV inactivating protein that can exist as a monomer or a domain-swapped dimer. We measured thermodynamic and kinetic parameters of the conversion process and determined the size of the energy barrier between the two species. The barrier is very large and of similar magnitude to that for equilibrium unfolding of the protein. Therefore, for CV-N, overall unfolding of the polypeptide is required for domain swapping.

Download full-text


Available from: Ivet Bahar
  • Source
    • "In the context of the broader 19 F protein NMR literature, these dynamical results on the NS1A ED dimer interface constitute a novel application of 19 F NMR relaxation to directly probe slow motion dynamics within a protein:protein interface. The NMR properties of 19 F have been exploited to illuminate slow motion conformational dynamic processes in other types of interactions, including protein:ligand interactions (Ahmed et al., 2007), peptide:bicelle interactions (Buer et al., 2010), changes in dynamics accompanying protein complex formation (Acchione et al., 2012), and the thermodynamic and kinetic aspects of domain swapping (Liu et al., 2012). In addition, 19 F CPMG relaxation approaches were recently used to characterize the conformational exchange behavior of fluorinated Phe residues in the thermal folding intermediate of calmodulin (Kitevski-Leblanc et al., 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nonstructural protein 1 of influenza A virus (NS1A) is a conserved virulence factor comprised of an N-terminal double-stranded RNA (dsRNA)-binding domain and a multifunctional C-terminal effector domain (ED), each of which can independently form symmetric homodimers. Here we apply (19)F NMR to NS1A from influenza A/Udorn/307/1972 virus (H3N2) labeled with 5-fluorotryptophan, and we demonstrate that the (19)F signal of Trp187 is a sensitive, direct monitor of the ED helix:helix dimer interface. (19)F relaxation dispersion data reveal the presence of conformational dynamics within this functionally important protein:protein interface, whose rate is more than three orders of magnitude faster than the kinetics of ED dimerization. (19)F NMR also affords direct spectroscopic evidence that Trp187, which mediates intermolecular ED:ED interactions required for cooperative dsRNA binding, is solvent exposed in full-length NS1A at concentrations below aggregation. These results have important implications for the diverse roles of this NS1A epitope during influenza virus infection.
    Full-text · Article · Feb 2014 · Structure
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: (R)- and (S)-Enantiomers of chiral metal free tetrakis(dinaphtho[1,2-e:1',2'-g]-1,4-dioxocine)-[2,3-b;2',3'-k;2'',3''-t; 2''',3'''-c']phthalocyanine (1) were synthesized via a cyclic tetramerization of the corresponding optically active benzo[b]dinaphtho[2,1-e:1',2'-g][1,4]dioxocine-5,6-dicarbonitrile (2) in refluxing n-pentanol in the presence of lithium followed by treatment with acetic acid. This novel chiral phthalocyanine compound has been characterized by a series of spectroscopic methods in addition to elemental analysis. The absolute molecular structures of both enantiomers have been unambiguously elucidated by single crystal X-ray diffraction analysis, resulting in the direct assignment of the chirality of metal free phthalocyanine 1.
    Preview · Article · Jul 2012 · Acta Chimica Sinica -Chinese Edition-
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The C-terminal domain (M(pro)-C) of SARS-CoV main protease adopts two different fold topologies, a monomer and a 3D domain-swapped dimer. Here, we report that M(pro)-C can reversibly interconvert between these two topological states under physiological conditions. Although the swapped α(1)-helix is fully buried inside the protein hydrophobic core, the interconversion of M(pro)-C is carried out without the hydrophobic core being exposed to solvent. The 3D domain swapping of M(pro)-C is activated by an order-to-disorder transition of its C-terminal α(5)-helix foldon. Unfolding of this foldon promotes self-association of M(pro)-C monomers and functions to mediate the 3D domain swapping, without which M(pro)-C can no longer form the domain-swapped dimer. Taken together, we propose that there exists a special dimeric intermediate enabling the protein core to unpack and the α(1)-helices to swap in a hydrophobic environment, which minimizes the energy cost of the 3D domain-swapping process.
    Full-text · Article · Aug 2012 · Proceedings of the National Academy of Sciences
Show more