Monitoring orientation and dynamics of membrane-bound melittin utilizing dansyl fluorescence.
Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. In spite of a number of studies, there is no consensus regarding the orientation of melittin in membranes. In this study, we used a melittin analogue that is covalently labeled at its amino terminal (Gly-1) with the environment-sensitive 1-dimethylamino-5-sulfonylnaphthalene (dansyl) group to obtain information regarding the orientation and dynamics of the amino terminal region of membrane-bound melittin. Our results show that the dansyl group in Dns-melittin exhibits red edge excitation shift in vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine, implying its localization in a motionally restricted region of the membrane. This is further supported by wavelength-dependent anisotropy and lifetime changes and time-resolved emission spectra characterized by dynamic Stokes shift, which indicates relatively slow solvent relaxation in the excited state. Membrane penetration depth analysis using the parallax method shows that the dansyl group is localized at a depth of approximately 18 A from the center of the bilayer in membrane-bound Dns-melittin. Further analysis of dansyl and tryptophan depths in Dns-melittin shows that the tilt angle between the helix axis of membrane-bound melittin and the bilayer normal is approximately 70 degrees. Our results therefore suggest that melittin adopts a pseudoparallel orientation in DOPC membranes at low concentration.
Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India.
Sourav
S
Haldar
H
H
Raghuraman
Amitabha
A
Chattopadhyay
D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000737:chemistry*; D008555:Melitten / Q000737:chemistry*; D015394:Molecular Structure; D013816:Thermodynamics; D013997:Time Factors
D005456:Fluorescent Dyes; D008555:Melitten