Conformational change of mastoparan from wasp venom on binding with phospholipid membrane.

The conformational change upon binding with phospholipid membrane has been studied of mastoparan from wasp venom, a tetradecapeptide causing the degranulation of mast cells. The 270-MHz 1H-NMR spectra and CD spectra indicate that the mastoparan molecule takes the alpha-helical conformation in methanol solution, but a much less ordered form in aqueous solution. On binding with phospholipid membrane, the alpha-helical conformation is formed even in aqueous medium. Such a conformational change is primarily due to the interaction between the aliphatic side chains of mastoparan and the hydrophobic interior of phospholipid membrane, in contrast to the case of melittin from bee venom.

Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogues by 1H nuclear magnetic resonance: correlation between activities and membrane-bound conformations.

Leu-enkephalin, [D-Ala2]Leu-enkephalin, and [D-Ala2]Leu-enkephalinamide (agonists) and [L-Ala2]Leu-enkephalin (inactive analogue) bind to lipid bilayer consisting of phosphatidylcholine and phosphatidylserine. The conformations that these compounds assume, once bound to perdeuterated phospholipid bilayer, have been shown to be unique, as shown by the transferred nuclear Overhauser effect (TRNOE) of 1H NMR spectroscopy. In addition, their location in the bilayer was analyzed by TRNOE in the presence of spin-labeled phospholipids. These analyses showed a clear relationship between the activity and the peptide-membrane interaction. The three active peptides, when bound to membranes, adopt the same conformation, characterized by a type II' beta-turn around Gly3-Phe4 and a gamma-turn around Gly2 (or D-Ala2). The inactive analogue, [L-Ala2]Leu-enkephalin, displayed a completely different TRNOE pattern corresponding to a different conformation in the membrane-bound state. The tyrosine residue of the active compounds is not inserted into the interior of membrane, but it is inserted into the bilayer for the L-Ala2 analogue. According to these results, [L-Ala2]Leu-enkephalin may be explained to be inactive because the mode of binding to the membranes is different from that of active compounds.

Vesicle-bound conformation of melittin: transferred nuclear Overhauser enhancement analysis in the presence of perdeuterated phosphatidylcholine vesicles.

We determined a detailed conformation of the honeybee venom peptide melittin when bound to phosphatidylcholine vesicles using proton NMR. In the presence of vesicles of perdeuterated dipalmitoylglycerophosphocholine, two-dimensional transferred nuclear Overhauser enhancement (TRNOE) experiments were carried out. By a distance geometry calculation using NOE-derived distance constraints followed by a simulated annealing refinement, the N-terminal (Leu6-Leu10) and C-terminal (Leu13-Lys21) parts were found to have an alpha-helical conformation, whereas five C-terminal residues (Arg22-Gln26) did not show a unique conformation in the vesicle-bound state. The two alpha-helices were connected via a less structured segment (Thr11-Gly12) with a helix bend angle of 86 degrees +/- 34 degrees. Model distance geometry calculations using distance constraints extracted from a tetrameric melittin molecule in crystal assured us that the NOE constraints can accurately reproduce melittin's structure, as well as helping to interpret the NMR structures. Although the vesicle-bound conformation of melittin is similar to that occurring in a methanol solution and in dodecylphosphocholine micelles, significant differences were found in the conformation of C-terminal basic residues and the helix bend angle. This is the first study to clearly demonstrate conformation differences in micelle- and vesicle-bound peptides. In addition, lytic activity of melittin and its analogs showed better correlation with a peptide conformation in vesicles than in either methanol or micelles.

The effect of activating ligands on the intrinsic fluorescence of guanine nucleotide-binding regulatory proteins.

The intensity of the tryptophan fluorescence of the alpha subunits of guanine nucleotide-binding regulatory proteins increases when they bind guanosine 5'-O-(3-thio)triphosphate (GTY gamma S). The kinetics of the fluorescence enhancement and of the measured binding of [35S]GTP gamma S are well correlated. The addition of Mg2+ to the nucleotide-bound proteins causes a further, rapid increase in the fluorescence intensity. Similar effects result from exposure of the proteins to F- and Mg2+, and the required concentration of F- is reduced by the inclusion of Al3+. It is presumed that the more highly fluorescent state of the G protein alpha subunits represents their active conformation.