Green
Fluorescent Protein 
Index 
Fluorescence Quenching Green
Fluorescent Protein
Index Fluorescence Quenching
The Local Environment of Tryptophan in Melittin
Melttin is a small amphipathic protein from bee venom, Apis mellifera which contains 26 amino acids and a single tryptophane residue. The sequence starts with 20 apolar residues and finishes with 4 charged and 2 polar residues. At neutral pH, melittin has a net charge of +5 to +6 . The carboxy terminus is amidated and thus neutral, which further enhances the positive charge of this peptide. Melittin is known to have cytolytic and voltage-gated ion conductivities. This toxic protein has been shown to integrate into natural or synthetic membranes and to disrupt a variety of cells and liposomes at micromolar concentrations. The fact that melittin can act as a lytic agent is partly due to its detergent-like sequence.
Melittin self-associates in solution. It can be a monomer, a dimer or a tetramer. In solutions with physiological pH, monomeric protein can be found in roughly equal proportions with tetrameric protein. The molecule is tetrameric at concentrations prevailing in the venom sac of the bee and monomeric at minimum concentrations required for cell lysis.
Melittin exists in a disordered state with low helicity in aqueous solutions. In the phospholipid bilayer, melittin is an alpha-helical monomer. When a voltage is applied across the membrane, four melittin molecules insert into bilayer to form a channel. The melittin helicity increases with channel formation which indicates bilayer induction of secondary structure. The presence of a distinct isodichroic point in the CD spectra of melittin demonstrates a two-state transition in which there are only two populated states: monomeric melittin in water with low helicity and membrane-bound melittin with high helicity. Thus folding appears very tightly coupled to partitioning in this protein.
Fluorescent Spectra
The spectral properties of the tryptophan residue are sensitive to both self-association and to the binding of melittiun to membranes. The sensitivity of tryptophane residues to the surrounding polarity allows for quantification of these phenomena.
The association of
melittin with itself and with lipid membranes illustrates typical
spectral changes found upon association. Self-association of
melittin to tetramers results in a blue shift of the tryptophane
emission spectrum (left). The self-association can
be induced by increasing the salt concentration. An increase in
melittin concentration also results in similar blue shifts.The
blue shifts are interpreted as a decrease in the exposure of the
single tryptophan residue upon formation of the teramer and a
stronger binding to the negatively charged lipid.
The fuorescence polarization and anisotropy of the tryptophan residue is also sensitive to the formation of tetramers. Upon assopciation, the polarization is increases from 0.07 to 0.12. The changes in polarization are primarily due to the increase in apparent volume of melittin upon self-association. These data can be used to quantify the association constant.
3-Dimensional Structure
The high
resolution structure of melittin has been solved first by X-ray
crystallography and then by NMR in methanol and in detergent
micelles. These structures share a common feature: alpha-helix as
shown left. Each melittin chain is composed of two alpha-helical
segments and its overall shape is that of a bent rod. Between the
two alpha-helical parts, there is a turn. The two NMR structures
are monomeric. In the crystal structure, melittin appears to form
a tetramer. This represents the self-associated form of melittin
at high ionic strength, high pH or high peptide concentration in
water.
The membrane channel that melittin forms, is a bundle of four parallel melittin monomer helices. The subunit-subunit contacts in the tetramer can be described in terms of helix-helix contacts. The hydrophobic residues on the surfaces of each chain are packed in the interior of the tetramer.Thus the interior is apolar and has no binding sites for the membrane. The hydrophilic face of each monomer points inwards to the lumen which allows melittin to bind to bilayers. The binding sites of melittin are formed by the side chains of the surface residues.
Click here for summaries and structural analyses of the melittin
files in the PDBsum database:
2mlt: melittin, X-ray 1bh1: d-pro melittin,
NMR
Then download the
coordinate file 2mlt for use in Rasmol. The file
contains the coordinates for the dimer in the
assymetrical unit. At the Rasmol command promt, type:
Examine the residue environment and exposure of the tryptophane residue in the graphic window.
