Abstract
Circular dichroism and tryptophan fluorescence spectroscopy have been used to investigate the structures of the influenza virus membrane glycoprotein hemagglutinin, acid-treated hemagglutinin, and fragments of hemagglutinin derived by proteolysis. The conformational change in hemagglutinin which occurs at the pH of membrane fusion (pH 5-6) was associated with a significant change of the environment of tyrosine residues, a change in the environment of tryptophan residues, but no changes in secondary structure. Tryptic digestion of the hemagglutinin in its low pH conformation which releases one of the subunit polypeptides (HA1) caused minimal changes in tyrosine and tryptophan environments but a small secondary structural change in HA1. The secondary structure of the remainder of the molecule (HA2) was very similar to that predicted from the known x-ray crystallographic structure of the native molecule. However, fluorescence spectroscopy indicated a tertiary change in structure in the coiled coil of alpha-helices which form the fibrous central stem of the molecule. These results are consistent with a conformational change required for membrane fusion which involves a decrease of HA1/HA1, HA1/HA2 interactions and changes in tertiary structure not accompanied by changes in secondary structure.
Highlights
From the Divisions of Virology and Physical Biochemistry, National Institute for Medical Research, TheRidgeway, Mill Hill, LondonNW7 MA, Great Britain and the $Department of Biochemistry and Molecular Biology, Harvard University, Cambridge,Massachusetts 02138
Since this is essentially identical to Secondary structure evaluated from far UV CD spectra compared the spectrum of BHA5, tryptic cleavage of BHA5 does not with estimates from the x-ray structure of BHA7 lead to substantial changes in tertiary structure detected by Comparison of the secondary structure derived from x-ray crystal- CD of the aromatic amino acids
7.70 strain, influenza virus hemagglutinins undergo conformational changes as a result of which the viruses can fuse with target membranes
Summary
Changes in structure in the coiled coil oaf-helices which form the the secondary and tertiary structureare discussed in relation fibrous central stem of the molecule These results atroe theknown structure of the molecule determined at neutral consistent with a conformational change required forpH. BHA-Hemagglutinin was isolated from virus by digestion with bromelain as previously described (Brand and Skehel, 1972). In the initialstages of infection, virus-receptor complexes are taken intoendosomes centrifugation, BHA was purified in a sucrose gradient as previously described (Skehel et al, 1982). The abbreviations used are: HA, hemagglutinin; BHA, bromelain- 5, cleavage at residue 224 does not occur and HA, 28-328 is released released hemagglutinin; PBS, phosphate-buffered saline; TBHA,, as a singlepolypeptide.
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