Conformational distributions of melittin in water / methanol mixtures from frequency-domain measurements of nonradiative energy transfer

Joseph R. Lakowicz, Ignacy Gryczynski, Wieslaw Wiczk, Gabor Laczko, Franklyn C. Prendergast, Michael L. Johnson

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Abstract

We used fluorescence energy transfer to examine the effects of solvent composition on the distribution of distances between the single tryptophan residue of melittin (residue 19) to the N-terminal α-amino group, which was labeled with a dansyl residue. The tryptophan intensity decays, with and without the dansyl acceptor, were measured by the frequency-domain method. The data were analyzed by a least-squares algorithm which accounts for correlation between the parameters. A wide distribution of tryptophan to dansyl distances was found for the random-coil state, with a Gaussian half-width of 25 Å. Increasing concentrations of methanol, which were shown to induce an α-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 Å at 80% (v/v) methanol. The distance from the indole moiety of Trp-19 to the dansyl group in 80% (v/v) methanol/water was found to be 25 Å, as assessed from the center of the distance distribution. A distance of 24-25 Å was recovered from the X-ray crystal structure of the tetramer, which is largely α-helical. At low ionic strength ( < 0.01) the CD spectra revealed a small fraction or amount of α-helix for molittin in water, which implies a small fraction of residual structure. This residual structure is apparently lost in guanidine hydrochloride as demonstrated by a further broadening in the distribution of distances. These results demonstrate the usefulness of frequency-domain measurements of resonance transfer for resolution of conformational distributions of proteins.

Original languageEnglish (US)
Pages (from-to)99-115
Number of pages17
JournalBiophysical Chemistry
Volume36
Issue number2
DOIs
StatePublished - 1990

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Keywords

  • Distance distribution
  • Energy transfer
  • Fluorescence spectroscopy
  • Frequency-domain fluorescence
  • Melittin
  • Time-resolved fluorescence

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Physical and Theoretical Chemistry

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