The biological importance of membrane protein is emphasized in statistical analyses of the genomes that indicateabout 30% of all open reading frames encode membrane proteins. For the purpose of performing structural biologyon membrane proteins, we have been developing a methodology for chemical synthesis of membrane proteins. Now,accumulated knowledge and experience has enabled us to work on the structure and function of transmembrane-jux-tamembrane region of membrane protein with solid state NMR and other spectroscopic techniques. Since littleattention has been paid to this portion of protein as such, obtaining the structural information thatprovides a clue tofully understand how membrane protein, especially, receptor on the membrane, passesthe structural change at theoutside to the inside. In this review, we introduce our approach from peptide chemistry to work on structural biolo-gy of membrane protein.

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The delivery of drugs involving bioactive peptides and proteins from water to natural cell membranes is crucial as aprimary step of the biological action. Lipid bilayer membrane, as a platform of vital functions, is a dynamic structurewhere molecules are always fluctuating under physiological conditions. The mechanism of drug deliveries is relatedto the molecular dynamics in such soft, fluid membrane interface. To gain insight into molecular mechanisms of drug deliveries in a noninvasive manner, we develop the method tomonitor dynamic properties of drugs and lipid components in membranes without labeled nuclei, by applying multi-nuclear high–resolution solution NMR in combination with the pulsed–field–gradient technique. The simultaneousobservation of membrane–bound and free drugs in situenables us to quantify i)in what amount the drug is bound insituand ii)how fast the drug is moving within the membrane, in relation to the thermal fluctuation of the soft, fluidenvironment. Finally, the method is extended to the in–cell NMR spectroscopy to detect the real–time uptake ofbioactive peptides with minimal perturbation.

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So far almost all studies of biomembrane and lipid membranes have been done using a suspension of manysmall–size vesicles such as large unilamellar vesicles (LUVs).In these studies, the average values of the physicalparameters of LUVs have been obtained from many LUVs, and thereby much information has been lost. Recently wehave proposed a novel method, the single GUV method, for probing function and dynamics of biomembranes/lipidmembranes. In this method we observe and measure changes of structures and physical properties of single giantunilamellar vesicles (GUVs)with a diameter of 10μm in the interaction with various substances, and analyzethese results over many “single GUVs”statistically. This method will reveal detailed elementary processes and alsodetermine their rate constants, which cannot be obtained by the conventional method.In this review, we describe anapplication of the single GUV method to studies on interactions of peptides with lipid membranes, especially interac-tion of antimicrobial peptide magainin 2 with lipid membranes. We show new valuable information on the magainin2–induced leakage of internal contents of liposomes and pore formation in lipid membranes. On the basis of thesedata, we can conclude that the magainin 2 concentration in the membrane interface determine the rate of the poreformation. We also discuss the advantage of the single GUV method in biomembrane research.

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Exchangeable apolipoproteins function in lipid transport as structural components of lipoprotein particles, cofac-tors for enzymes and ligands for cell–surface receptors. Recent findings with apolipoproteins A–I and E suggest thatthe tertiary structures of these two members of the human exchangeable apolipoprotein gene family are related.Characteristically, these proteins contain a series of proline–punctuated, 11–or 22–amino acid, amphipathicα–heli-cal repeats that can adopt a helix bundle conformation in the lipid–free state. The amino–and carboxyl–terminalregions form separate domains with the latter being primarily responsible for lipid binding. Interaction with lipidinduces changes in the conformation of the amino–terminal domain leading to alterations in function. The concept ofa two–domain structure for the larger exchangeable apolipoproteins is providing new molecular insights into howthese apolipoproteins interact with lipids on cell surfaces and lipoprotein particles.

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Although, studies of lipid metabolism have been focusing on that in the systemic circulation using nonneuronalcells for decades, knowledge regarding cholesterol metabolism in the brain, the most lipid–rich organs, is very limit-ed. The cholesterol metabolism in the brain is supposed to be independently and uniquely regulated, because brainis segregated from the systemic circulation by the blood–brain barrier. Thus, the cholesterol being transported inthe systemic circulation by serum lipoprotein cannot be available to the brain.It has been shown that cholesterolsupplied as a lipoprotein complex, i.e., high–density–lipoprotein (HDL), is critical for the maturation of synapses andthe maintenance of synaptic plasticity.Interestingly, apolipoprotein E (apoE)is the major apolipoprotein generatingHDL in the brain, which is supplied to neurons via apoE receptors. The discovery that possession of apoE alleleε4is a strong risk factor for Alzheimer’s disease (AD)leads us to focus on the role of cholesterol in the pathogenesis ofAD. Accumulating epidemiological and biological evidence suggests the link between the serum cholesterol leveland the development of AD, and the potential therapeutic effectiveness of statins for AD and mild cognitive impair-ment (MCI), whereas other lines of evidence show controversial results. Cholesterol is known to interact with amy-loidβ–protein (Aβ)in a reciprocal manner: cellular cholesterol levels modulate Aβgeneration, while, Aβalters cho-lesterol dynamics in neurons, leading to tauopathy.In this review, the relationship between the cholesterol levels inserum or cerebrospinal fluid (CSF)and the induction of AD is discussed. The mechanism(s), if this is the case, ofhow cholesterol in the central nervous system (CNS)is involved in the induction of pathologies of AD includingAβgeneration and tauopathy, and how statins prevent it are also discussed.

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The role of liposome addition on the gene expression of green fluorescent protein (GFP)was studied in an in vitroprotein translation system in the presence of cationic liposome, prepared by 1,2–dioleoyl–3–trimethyl–ammonium–propane (DOTAP). GFP fluorescence after gene expression was reduced with an increase in DOTAP concentrations,and no fluorescence was observed at certain concentrations of DOTAP. It was found that neither translated GFP nortranscript mRNA for GFP were produced at the above conditions. The above results may show that the DOTAP lipo-some canaffect the inhibition at the transcription step of the GFP synthesis process in addition to its translation andfolding steps.

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In human erythrocyte membranes, multidrug resistance–associated protein (MRP) 1, 4 and 5 are expressed. Inthis study, transport function of MRP5 was evaluated using 5–(and–6)–carboxy–2’, 7’dichlorofluorescein (CDCF) asa substrate. The uptake of CDCF by inside–out vesicles (IOVs) prepared from human erythrocytes was anATP–dependent, active process, and was saturable with a Km value of 32.9μM. CDCF uptake was almost complete-ly inhibited by probenecid and sulfinpyrazone. On the other hand, prostaglandin E1(PGE1), an inhibitor of MRP4,did not affect CDCF uptake, though the uptake of methotrexate (MTX) by IOVs was strongly inhibited by PGE1.Similarly, cGMP and panipenem, a carbapenem antibiotic, inhibited MTX but not CDCF uptake, while valproic acidinhibited the uptake of both compounds. These results indicate that CDCF is a suitable compound to analyze MRP5function in human erythrocyte membranes, and sensitivity to several compounds such as PGE1, cGMP and panipen-em is different between MRP5 and MRP4.

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A new cosmetic ASPION CE was commercialized by utilizing a novel surfactant coating technique for L–ascorbicacid phosphate magnesium salt (Vitamin C).The coating technique can change the surface properties of cosmeticingredients. In developing the ASPION CE, the hydrophilic property of ascorbic acid derivative was converted to thehydrophobic one, which enables to dissolve it in natural oils. As the result, we can completely dissolve the coatedVitamin C and the hydrophobic Vitamin E in an oil phase, and can get a homogeneous cosmetic oil. The particlesizes can be controlled around several hundreds nanometer by changing preparation conditions. The oil–based cos-metic was available for carrying the Vitamin C to the melanosite through the cutaneous tissue, because the outer skinis hydrophobic. Furthermore, immobilized Vitamin C was stabilized by coating with the surfactant layer.

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