Methods for studying transmembrane peptides in bicelles: consequences of hydrophobic mismatch and peptide sequence.

We have shown that bicelles prepared from dilauryl phosphatidylcholine (DLPC) and dipalmitoyl phosphatidylcholine (DPPC) align in a magnetic field under conditions similar to the more common dimyristoyl phosphatidylcholine (DMPC) bicelles. In addition, a model transmembrane peptide, P16, with a hydrophobic stretch of 24 A, and specific alanine-d(3) labels, was incorporated into all of the different bicelles. The long-chain phospholipid (DLPC, DMPC, or DPPC) remained unperturbed upon incorporation of the peptide while the quadrupolar splitting of the short-chain phospholipid along the bicelle rim increased by varying degrees in the different bicelle systems. The change in quadrupolar splitting of the short-chain phospholipids was attributed to changes in either fluidity of the planar region of the bicelle or differences in overall lipid packing. When the hydrophobic stretch of the bilayer was 22.8 (DMPC) or 26.3 A (DPPC), the peptide tilt was found to be transmembrane (33-35 degrees with respect to the bicelle normal). When the hydrophobic stretch of the bilayer was 19.5 A (DLPC), the peptide quadrupolar splittings suggested a loss of transmembrane orientation. When tryptophan was incorporated in the middle of the transmembrane region, the transmembrane orientation was also lost.

Position of residues in transmembrane peptides with respect to the lipid bilayer: a combined lipid Noes and water chemical exchange approach in phospholipid bicelles.

The model transmembrane peptide P16 (Ac-KKGLLLALLLLALLLALLLKKA-NH2) was incorporated into small unaligned phospholipid bicelles, which provide a 'native-like' lipid bilayer compatible with high-resolution solution NMR techniques. Using amide-water chemical exchange and amide-lipid cross-relaxation measurements, the interactions between P16 and bicelles were investigated. Distinctive intermolecular NOE patterns observed in band-selective 2D-NOESY spectra of bicellar solutions with several lipid deuteration schemes indicated that P16 is preferentially interacting with the 'bilayered' region of the bicelle rather than with the rim. Furthermore, when amide-lipid NOEs were combined with amide-water chemical exchange cross-peaks of selectively 15N-labeled P16 peptides, valuable information was obtained about the position of selected residues relative to the membrane-water interface. Specifically, three main classes were identified. Class I residues lie outside the bilayer and show amide-water exchange cross-peaks but no amide-lipid NOEs. Class II residues reside in the bilayer-water interface and show both amide-water exchange cross-peaks and amide-lipid NOEs. Class III residues are embedded within the hydrophobic core of the membrane and show no amide-water exchange cross-peaks but strong amide-lipid NOEs.

Bicelles in structure-function studies of membrane-associated proteins.

Bicelles are a novel form of long-chain/short-chain phospholipid aggregates, which are useful for biophysical and biochemical studies of membrane-associated biomolecules. In this work, we review the development of bicelles and their uses in structural characterization (primarily via NMR, circular dichroism, and fluorescence) of membrane-associated peptides. We also show that bicellar phospholipids are substrates for lipolytic enzymes. For this latter work, we employed a 31P NMR enzymatic assay system to examine the kinetic behavior of cobra venom phospholipase A(2) toward a variety of bicellar substrates. This enzyme hydrolyzed all bicelle lipids at rates comparable to those found for the enzyme action on traditional micellar substrates, which are the best substrates for this enzyme. In addition, we found that this PLA(2) showed no significant preference for long-chain or short-chain phospholipids when they were presented as mixtures in bicelles.