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Melittin-phospholipid interaction studied by employing the single tryptophan residue as an intrinsic fluorescent
Abstract:
The rotational correlation time of melittin, obtained from the nanosecond anisotropy of the emission from its single tryptophan residue, has been found to increase considerably in phosphate solution relative to that in aqueous solution, consistent with protein aggregation. The steady-state fluorescence spectra as well as the absorption spectra in phosphate solution exhibit a very good degree of similarity with those of the protein bound to egg phosphatidylcholine (PC) and distearoylphosphatidylcholine (DSPC) bilayer liposomes. The value of the second-order rate constant for dynamic quenching, kq = 1.4.10(9) M-1.s-1, by acrylamide in 0.5 M phosphate solution is comparable to those for the protein-phospholipids complexes (1.10(9) and 0.7.10(9) M-1.s-1 for egg PC and DSPC, respectively). Similarities are also found in the nanosecond properties. There is a much stronger and quite similar dependence of the fluorescence spectra on time in the nanosecond range and of the fluorescence decay times on the emission wavelength in both cases as compared to the case is aqueous solution. These observations support the notion that melittin binds to the phospholipids in an aggregated form. The results suggest that the reduction in the kq values of bound melittin relative to that in aqueous solution and the blue shift of the fluorescence spectrum (from 352 to 337 nm) are brought about by shielding of the tryptophan residue from the solvent through a combination of protein aggregation and enhancement of its alpha-helical content (suggested by published CD data). The magnitude of the kq values for bound melittin, however, is still relatively high implying the occurrence of rather frequent encounters between the tryptophan residue and the hydrophilic acrylamide molecules. Thus, the residue is found not to penetrate deep into the phospholipid bilayer.
Insights
Melittin protein aggregation in phosphate solutions increases its rotational correlation time. This behavior, similar to melittin bound to liposomes, suggests aggregation influences its interaction with phospholipids.
Area of Science:
- Biophysics
- Protein Chemistry
- Spectroscopy
Background:
- Melittin, a peptide toxin, is known to interact with lipid bilayers.
- Understanding melittin's aggregation state is crucial for elucidating its biological activity and membrane interactions.
Purpose of the Study:
- To investigate the aggregation state of melittin in phosphate solutions.
- To compare melittin's behavior in phosphate solutions with its interaction with phospholipid bilayers.
Main Methods:
- Nanosecond fluorescence anisotropy measurements to determine rotational correlation time.
- Steady-state absorption and fluorescence spectroscopy.
- Dynamic quenching studies using acrylamide.
Main Results:
- Rotational correlation time of melittin significantly increased in phosphate solution, indicating protein aggregation.
- Spectroscopic properties (absorption and fluorescence) in phosphate solution closely resembled those of melittin bound to phosphatidylcholine (PC) and distearoylphosphatidylcholine (DSPC) liposomes.
- Dynamic quenching rate constants (kq) by acrylamide were comparable between melittin in phosphate solution and melittin bound to liposomes.
- Fluorescence spectra and decay times showed similar time- and wavelength-dependent behaviors in phosphate solution and when bound to liposomes, distinct from aqueous solution.
Conclusions:
- Melittin binds to phospholipids in an aggregated form in phosphate solutions.
- Protein aggregation and increased alpha-helical content shield the tryptophan residue, reducing kq values and causing a blue shift in fluorescence.
- The tryptophan residue does not deeply penetrate the phospholipid bilayer, as indicated by the relatively high kq values.