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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Carrier Proteins / chemical synthesis
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Carrier Proteins / genetics
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Carrier Proteins / ultrastructure
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Detergents / chemistry
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Enzyme-Linked Immunosorbent Assay
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Escherichia coli Proteins / chemical synthesis
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / ultrastructure
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Genetic Vectors
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Hemagglutinin Glycoproteins, Influenza Virus / biosynthesis
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Hemagglutinin Glycoproteins, Influenza Virus / chemistry*
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Hemagglutinin Glycoproteins, Influenza Virus / genetics
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Hemagglutinin Glycoproteins, Influenza Virus / ultrastructure
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Hemagglutinins, Viral / biosynthesis
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Hemagglutinins, Viral / chemistry*
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Hemagglutinins, Viral / genetics
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Hemagglutinins, Viral / ultrastructure
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Hydrogen-Ion Concentration
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Maltose-Binding Proteins
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Molecular Sequence Data
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Protein Conformation
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Protein Folding*
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Recombinant Fusion Proteins / chemical synthesis
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / ultrastructure
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Solubility
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Structure-Activity Relationship
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Ultracentrifugation
Substances
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Carrier Proteins
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Detergents
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Escherichia coli Proteins
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Hemagglutinin Glycoproteins, Influenza Virus
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Hemagglutinins, Viral
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Maltose-Binding Proteins
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Recombinant Fusion Proteins
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hemagglutinin HA-2 fusogenic peptide, Influenza virus