Distributions of fluorescence decay times for synthetic melittin in water-methanol mixtures and complexed with calmodulin, troponin C, and phospholipids

Joseph R. Lakowicz, Ignacy Gryczynski, Wieslaw Wiczk, Michael L. Johnson

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Abstract

Frequency-domain measurements of the intensity decays of melittin were used to recover the distribution of decay times displayed by its single tryptophan residue. Melittin was examined in the monomeric random coil state (water), in the monomeric α-helical state (water-methanol), in the tetrameric state, and with 6 M guanidine hydrochloride. In the presence of denaturant, where melittin is expected to be devoid of secondary structure, we observed a narrow distribution of lifetimes, similar to a double-exponential decay. In water the intensity decay of melittin was found to be described better by the distribution of decay times, which became progressively wider as the amount of α-helix was increased by the methanol cosolvent or upon formation of the α-helical tetrameric state. We also examined the intensity decays of melittin when complexed with calmodulin, troponin C, or lipid vesicles of 1-palmitoyl-2-oleyl-l-α-phosphatidylcholine (POPC). The lifetime distributions of the complexes with lipid were comparable to those observed in methanol-water, suggesting a similarity of the structure and/or dynamics of the environment surrounding the tryptophan residue. A broad lifetime distribution was observed for the melittin-calmodulin complex, suggesting a rigid structure and/or heterogeneity in the form of the complex. The lifetime distribution of the melittin-troponin C complex was more narrow, suggesting a more uniform structure, at least in the region surrounding the tryptophan residue. These results demonstrate that the lifetime distributions of a single tryptophan protein can be a sensitive indicator of the conformational heterogeneity and dynamics of proteins.

Original languageEnglish
Pages (from-to)169-177
Number of pages9
JournalJournal of Fluorescence
Volume4
Issue number2
DOIs
StatePublished - Jun 1994

Keywords

  • Melittin
  • calmodulin
  • fluorescence decay times
  • phospholipids
  • troponin C
  • water-methanol

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