Abstract
To investigate the dynamics and conformation of the membrane-interacting HA2 domain of the hemagglutinin protein of influenza virus, the peripheral part of the HA2 domain (aa 1-127) was expressed in Escherichia coli. Four consecutive single-cysteine mutants, F63C, H64C, Q65C, and I66C, were generated using site-directed mutagenesis. This region is proposed to undergo a conformational change from a loop to a helical coiled-coil when going from the native to the fusion-active state [Bullough et al. (1994) Nature (London) 371, 37-43]. In the trimeric coiled-coil geometry positions 63 and 66 belong to the core so that cysteines from individual monomers are spatially close. On the other hand, positions 64 and 65 face the aqueous phase so that cyteines from monomers are spatially remote. The mutants were studied with cysteine-cysteine cross-linking and the spin-labeling electron paramagnetic resonance (EPR) in both the membrane-bound state and in the detergent-solubilized state. Extensive intramolecular cysteine-cysteine cross-linking was observed not only for F63C and I66C but also for H64C. Rates of cross-linking were comparable for these three mutants at physiological temperatures. These results are inconsistent with what is expec...Continue Reading
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