The interaction between receptors and guanine nucleotide binding (G) proteins leads to G protein activation and subsequent regulation of effector enzymes. The molecular basis of receptor-G protein interaction has been examined by using the ability of the G protein from rods (transducin) to cause a conformational change in rhodopsin as an assay. Synthetic peptides corresponding to two regions near the carboxyl terminus of the G protein alpha subunit, Glu311-Val328 and Ile340-Phe350, compete with G protein for interaction with rhodopsin. Amino acid substitution studies show that Cys321 is required for this effect. Ile340-Phe350 and a modified peptide, acetyl-Glu311-Lys329-amide, mimic G protein effects on rhodopsin conformation, showing that these peptides bind to and stabilize the activated conformation of rhodopsin.

Specular reflection of neutrons has been used to characterize the structure of single lipid bilayers adsorbed to a planar silicon surface from aqueous solution. We used a novel experimental setup which significantly decreased the incoherent background scattering and allowed us to measure neutron reflectivities as low as 5 × 10-7. Thicknesses and neutron scattering length densities were determined by a fitting procedure using (i) randomly generated smooth functions represented by parametric B-splines and (ii) stepped functions based on the theoretical lipid composition. The size of lipid domains at the surface and the degree of surface coverage were determined by atomic force microscopy. Chain-protonated and -deuterated dipalmitoylphosphatidylcholine (DPPC) bilayers were investigated in 2H2O and a mixture of 2H2O and H2O which matches the scattering density of silicon. Also, one measurement on a distearoylphosphatidylcholine bilayer which has longer acyl chains was performed for comparison. The lipid adsorbs to the silicon surface as a continuous layer interrupted by irregularly shaped uncovered areas which are 100−500 Å in size. The surface coverage was estimated to be 70 ± 20%. The reflectivity measurements on DPPC at 60 °C show a silicon oxide layer with a thickness of the order of 4 Å, a rough silicon oxide/water layer between silicon oxide and lipid with a thickness between 2 and 8 Å, and a single lipid bilayer. Fitting resolved a central membrane layer with a thickness of 28 ± 2 Å which represents the lipid hydrocarbon chains. This layer is sandwiched between interface membrane layers of lipid head groups and water which are 11.5 ± 1 Å in thickness. The angstrom-scale thickness changes of the central membrane layer as a function of the phase state of the lipid and of the length of the hydrocarbon chains are easily detected.

The elastic area compressibility modulus, Ka, of lamellar liquid crystalline bilayers was determined by a new experimental approach using 2H-NMR order parameters of lipid hydrocarbon chains together with lamellar repeat spacings measured by x-ray diffraction. The combination of NMR and x-ray techniques yields accurate determination of lateral area per lipid molecule. Samples of saturated, monounsaturated, and polyunsaturated phospholipids were equilibrated with polyethylene glycol (PEG) 20,000 solutions in water at concentrations from 0 to 55 wt % PEG at 30 degrees C. This procedure is equivalent to applying 0 to 8 dyn/cm lateral pressure to the bilayers. The resulting reductions in area per lipid were measured with a resolution of +/-0.2 A2 and the fractional area decrease was proportional to applied lateral pressure. For 1,2-dimyristoyl(d54)-sn-glycero-3-phosphocholine, 1-stearoyl(d35)-2-oleoyl-sn-glycero-3-phosphocholine (SOPC-d35), and 1-stearoyl(d35)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC-d35) cross-sectional areas per molecule in excess water of 59.5, 61.4, and 69.2 A2 and bilayer elastic area compressibility moduli of 141, 221, and 121 dyn/cm were determined, respectively. Combining NMR and x-ray results enables the determination of compressibility differences between saturated and unsaturated hydrocarbon chains. In mixed-chain SOPC-d35 both chains have similar compressibility moduli; however, in mixed-chain polyunsaturated SDPC-d35, the saturated stearic acid chain appears to be far less compressible than the polyunsaturated docosahexaenoic acid chain.

The immunophilin-like FKBP42 TWISTED DWARF1 (TWD1) has been shown to control plant development via the positive modulation of ABCB/P-glycoprotein (PGP)-mediated transport of the plant hormone auxin. TWD1 functionally interacts with two closely related proteins, ABCB1/PGP1 and ABCB19/PGP19/MDR1, both of which exhibit the ability to bind to and be inhibited by the synthetic auxin transport inhibitor N-1-naphylphtalamic acid (NPA). They are also inhibited by flavonoid compounds, which are suspected modulators of auxin transport. The mechanisms by which flavonoids and NPA interfere with auxin efflux components are unclear. We report here the specific disruption of PGP1-TWD1 interaction by NPA and flavonoids using bioluminescence resonance energy transfer with flavonoids functioning as a classical established inhibitor of mammalian and plant PGPs. Accordingly, TWD1 was shown to mediate modulation of PGP1 efflux activity by these auxin transport inhibitors. NPA bound to both PGP1 and TWD1 but was excluded from the PGP1-TWD1 complex expressed in yeast, suggesting a transient mode of action in planta. As a consequence, auxin fluxes and gravitropism in twd1 roots are less affected by NPA treatment, whereas TWD1 gain-of-function promotes root bending. Our data support a novel model for the mode of drug-mediated P-glycoprotein regulation mediated via protein-protein interaction with immunophilin-like TWD1.Bioactive flavonoids derived from plant secondary metabolism serve as important nutraceuticals (1). They have healthpromoting effects, including antioxidant, anticarcinogenic, antiviral, and anti-inflammatory activities; however, the cellular targets of the in vivo protein remain largely unknown (1, 2). In plants, among other functions, flavonoids such as quercetin, kaempferol, and other aglycone molecules have been shown to inhibit cell-to-cell/polar auxin transport (PAT) 3 and consequently to enhance localized auxin accumulation (1, 3-6). During PAT, the plant hormone auxin, which determines many aspects of plant physiology and development, is moved directionally in a cell-to-cell mode (7-9).The regulatory impact of flavonoids on PAT initially was based on their ability to compete with N-1-naphtylphtalamic acid (NPA), a synthetic auxin transport inhibitor (ATI) (4, 10 -12) and herbicide (naptalam, alanap), for transporter binding sites. This concept is further supported by auxin-related phenotypes of Arabidopsis mutants with altered flavonoid levels (1, 3, 13), although fundamental physiological processes occur in the absence of flavonoids. Currently the flavonoids are seen as transport regulators or modulators (14); nevertheless, the mechanisms by which flavonoids interfere with auxin efflux components are not yet clear (1).The auxin efflux complex is thought to regulate PAT on the molecular level and consists of at least two proteins: a membrane integral transporter and an NPA-binding protein (NBP) regulatory subunit (11,(15)(16)(17). Recently, ABCB/P-glycoprotein (PGP)/multidrug resistance (MDR) proteins, members o...