NMR studies of membrane-binding peptides in monoolein cubic phases

Membrane proteins are of particular interest to structural biologists since it is believed that these proteins comprise approximately thirty percent of the proteins encoded for by genomes. Despite the biological significance of these proteins, the atomic reso1utio:n structures of membrane proteins are being solved at a much slower rate than their soluble counterparts. This is primarily due to difficulties that arise in maintaining the lipid interactions required to retain the structural and functional integrity of the proteins during structural studies. One: approach to resolving this problem would be to develop alternative membrane-mimetic environments for structural studies of membrane proteins. The cubic phases formed by mixtures of lipids and water have been identified as such an environment. In recent years cubic phases have been used to crystallize membrane proteins for structural studies using X-ray crystallographic techniques. This application of cubic phases, along with information provided from previous studies, indicated that cubic phases possessed the necessary properties to make them ideal membrane-mimetic environments for solution NMR studies of membrane proteins. I investigated the suitability of the cubic phases formed by mixtures of monoolein and water as membrane-mimetic environments for the solution NMR study of incorporated membrane proteins. For my studies I used two transmembrane peptides (WNALAAVMAAVA&AAGKSKSKS and alamethicin), and one membrane surface-associating peptide (methionine-enkephalin), as models of membrane proteins. In the NMR spectra collected on the transmembrane peptides, only the residues found in the interfacial regions of the cubic phase were observed, whereas all of the residues of the membrane surface-associating peptide were observed. In order to gain insights into the behaviour of the incorporated peptides, the diffusion rates of lipid, peptide and water molecules were measured in peptide-containing cubic phases using :solution NMR techniques. The data that I have collected provide the first examples of solution 'NMR spectra collected on peptides incorporated into a lipid cubic phase. The NMR spectra that were obtained suggest that the monoo1ein:water cubic phase may be a suitable membrane-mimetic environment for the study of membrane surface-associating peptides and proteins, and the inter-helical loops of integral membrane proteins.