On the nature of interaction between melittin of bee venom and model phospholipid membranes
Date Issued
December 1, 1980
Author(s)
Thompson, Michael L.
Advisor(s)
Solon Georghiou
Abstract
The single tryptophan residue of melittin, the major component of bee venom, has been utilized as an intrinsic fluorescence probe for investigating the nature of binding of melittin to model phospholipid membranes. A recent CD study showed that 0.15 M inorganic phosphate increases 1 the α-helical content of melittin. The maximum of the fluorescence spectrum in 0.01 M Tris buffer (pH 7.0) is at 352 nm, virtually identical to that of free tryptophan in that buffer. Thus, the tryptophan residue is almost completely exposed to water. The spectrum blue-shifts to 336 nm upon addition of 0.5 M inorganic phosphate implying that the environment of the tryptophan residue becomes more hydrophobic. One molar NaCl causes similar results. The spectra for melittin are very similar with those in salt solutions. That is also the case for the absorption spectra that shift to the red by about 2 nm relative to that of free melittin. Free melittin has been found to exhibit very small time-resolved spectral shifts on the ns time scale and dependence of the fluorescence decay time on the emission wavelength resembling those exhibited by free tryptophan. For salt solutions and membranes, however, the effects are much more pronounced. The rotational correlation time, obtained from ns fluorescence depolarization measurements, increases from 1.1 ns for free melittin to 3.5 ns for 0.5 M K2HPO4, implying a different conformation in the salt solution. These findings can be rationalized in terms of aggregation of the protein in salt solutions and when bound to membranes in line with the suggested in the literature tetrameric form of the protein at high ionic strength. Acrylamide quenching measurements indicate that the tryptophan residue is much less exposed to the solvent when in salt solutions and when bound to membranes than in Tris buffer. In all cases, fluorescence quenching occurs by both diffusion-controlled and instantaneous processes. It is inferred that the tryptophan residue does not penetrate into the hydrophobic core of the membranes, which contradicts published reports.
Degree
Master of Science
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Thesis80T465.pdf
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