title: Full-length influenza hemagglutinin HA2 refolds into the trimeric low-pH-induced conformation.
abstract: The influenza virus uses hemagglutinin (HA) to fuse the viral and cellular membranes. As part of an effort to study the membrane-interacting elements of HA, the fusion peptide, and the C-terminal transmembrane anchor, we have expressed in Escherichia coli the full-length HA(2) chain with maltose-binding protein fused at its N-terminus. The chimeric protein can be refolded in vitro in the presence of specific detergents to yield stable, homogeneous trimers, as determined by analytical ultracentrifugation. The trimers have the so-called "low pH" conformation-the rearranged HA(2) conformation obtained when intact HA(1)/HA(2) is induced to refold by exposure to low pH-as detected by electron microscopy and monoclonalantibody reactivity. These results provide further evidence for the notion that the neutral-pH structure of intact HA is metastable and that binding of protons lowers the kinetic barriers that prevent rearrangement to the minimum-free-energy conformation. The refolded chimeric protein described here is a suitable species for undertaking studies of how the fusion peptide inserts into membranes and assessing the nature of possible intermediates in the fusion process.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Howard Hughes Medical Institute, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115, USA. swalley@crystal.harvard.edu
authors.forename: Susanne E
authors.initials: SE
authors.lastname: Swalley
authors.forename: Brian M
authors.initials: BM
authors.lastname: Baker
authors.forename: Lesley J
authors.initials: LJ
authors.lastname: Calder
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Harrison
authors.forename: John J
authors.initials: JJ
authors.lastname: Skehel
authors.forename: Don C
authors.initials: DC
authors.lastname: Wiley
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000648:ultrastructure; D003902:Detergents / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D029968:Escherichia coli Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000648:ultrastructure; D005822:Genetic Vectors; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D006389:Hemagglutinins, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000648:ultrastructure; D012995:Solubility; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation
chemical_list: D002352:Carrier Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; C452559:hemagglutinin HA-2 fusogenic peptide, Influenza virus