Intrinsic membrane proteins represent a large fraction of the proteins produced by living organisms and perform many crucial functions. Structural and functional characterization of membrane proteins generally requires that they be extracted from the native lipid bilayer and solubilized with a small synthetic amphiphile, for example, a detergent. We describe the development of a small molecule with a distinctive amphiphilic architecture, a “tripod amphiphile,” that solubilizes both bacteriorhodopsin (BR) and bovine rhodopsin (Rho). The polar portion of this amphiphile contains an amide and an amine-oxide; small variations in this polar segment are found to have profound effects on protein solubilization properties. The optimal tripod amphiphile extracts both BR and Rho from the native membrane environments and maintains each protein in a monomeric native-like form for several weeks after delipidation. Tripod amphiphiles are designed to display greater conformational rigidity than conventional detergents, with the long-range goal of promoting membrane protein crystallization. The results reported here represent an important step toward that ultimate goal.

Rice (Oryza sativa) plants were grown with their roots sandwiched between thin layers of phosphorus-deficient soil from which they were separated by fine mesh, and root-induced changes in the soil affecting phosphate solubility were measured. The concentrations of low molecular weight organic anions in the thin layers, particularly citrate, increased in the presence of the plants. Apparent rates of citrate excretion from the roots, calculated from the quantities in the soil and rates of decomposition calculated with a first order rate constant measured independently, varied from 337–155 nmol g−1 root f. wt h−1 over the course of plant growth, equivalent to 2–3% of plant d. wt. Rates of excretion were similar for NH4+ and NO3−-fed plants. The soil pH decreased from its initial value by up to 0.6 units for the NH4+-fed plants and increased by up to 0.4 units for the NO3−-fed ones. The contribution of organic anion excretion to the pH changes was small compared with that of the inorganic cation-anion balance in the plants. The extent to which the observed excretion of citrate and root-induced pH changes could account for the observed phosphate solubilization and uptake was assessed using a mathematical model. Previous work had shown that phosphate solubilization by rice in this soil could not be explained by enhanced phosphatase activity in the rhizosphere, and the roots were not infected with mycorrhizas. The model allows for the diffusion of the solubilizing agent (citrate or H+) away from the roots, its decomposition by soil microbes (citrate only); its reaction with the soil in solubilizing phosphate and diffusion of the solubilized phosphate to the roots. The model contains no arbitrary assumptions and uses only independently measured parameter values. The agreement between the measured time course of phosphorus uptake and that predicted for solubilization by citrate was good. Root-induced acidification by NH4+-fed plants resulted in additional solubilization, the acidification enhancing the solubilizing effect of citrate. However, the final phosphorus uptake by NH4+-fed plants was no greater than that of NO3−-fed plants, presumably because the acidification inhibited plant growth. The mechanism of solubilization by citrate involved formation of soluble metal-citrate chelates rather than displacement of phosphate from adsorption sites.