Metal binding to cowpea chlorotic mottle virus using terbium(III) fluorescence

Abstract

Metals are thought to play a role in the structure of many viruses. The crystal structure of the T=3 icosahedral cowpea chlorotic mottle virus (CCMV) suggests the presence of 180 unique metal-binding sites in the assembled protein cage. Each of these sites is thought to involve the coordination of the metal by five amino acids contributed from two adjacent coat protein subunits. We have used fluorescence resonance energy transfer (FRET), from tryptophan residues proximal to the putative metal-binding sites, to probe Tb(III) binding to the virus. Binding of Tb(III) was investigated on the wild-type virus and a mutant where the RNA binding ability of the virus was removed. Tb(III) binding was observed both in the wild-type virus (K d=19 μM) and the mutant (K d=17 μM), as monitored by the increase in Tb(III) fluorescence (545 nm) and concomitant decrease in tryptophan fluorescence (342 nm). Competitive binding experiments showed Ca(II) to have about 100-fold less affinity for the binding sites (K d=1.97 mM). This is the first direct evidence of metal binding to the putative metal-binding sites, originally suggested from the crystal structure of CCMV.

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Acknowledgements

This work was supported in part by the National Institutes of Health (RO1 GM61340) and the Office of Naval Research.

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Correspondence to Trevor Douglas.

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Basu, G., Allen, M., Willits, D. et al. Metal binding to cowpea chlorotic mottle virus using terbium(III) fluorescence. J Biol Inorg Chem 8, 721–725 (2003). https://doi.org/10.1007/s00775-003-0470-7

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Keywords

  • Cowpea chlorotic mottle virus
  • Fluorescence resonance energy transfer
  • Metal–RNA binding
  • Metal–virus binding
  • Terbium