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Biomedical subjects

W L Hubbell

Publications and source records attributed to W L Hubbell.

At least 109 records · Page 6Linked to original sources
PubMed1986

Hydrophobic ion interactions with membranes. Thermodynamic analysis of tetraphenylphosphonium binding to vesicles.

The thermodynamic properties for the interaction of the hydrophobic ion tetraphenylphosphonium (TPP+) with egg phosphatidylcholine vesicles were studied in detail by equilibrium dialysis and spin label techniques. A partition coefficient of beta = 4.2 + 0.4 x 10(-6) cm (K congruent to 100) was determined. Electrostatic saturation sets in at approximately 600 microM (about one absorbed TPP+ molecule per 100 lipids), and is not screened by salt. The temperature dependence of binding was determined, which reveals that the binding is entropy-driven with a positive (repulsive) enthalpy of binding, a result to be compared with hydrophobic anions in which the binding enthalpy is negative. The membrane dipole potential may be responsible for this binding difference. Activity coefficients are determined and shown to be significantly different from those of most common salts, an important result that should be considered in all hydrophobic ion studies. Comparison of the TPP+ results with those of its anionic structural analogue, tetraphenylboron (TPB-), permits a general analysis of hydrophobic ion interactions with membranes. A theoretical model consistent with the entire set of data is developed in an accompanying article.

Animals↗
PubMed1986

The membrane dipole potential in a total membrane potential model. Applications to hydrophobic ion interactions with membranes.

The total potential energy profile for hydrophobic ion interactions with lipid bilayers can be written as the sum of four terms: the electrical Born, image and dipole contributions, and a neutral energy term. We introduce a specific model for the membrane dipole potential, treating it as a two-dimensional array of point dipoles located near each membrane-water interface. Together with specific theoretical models for the other energy terms, a total potential profile is developed that successfully describes the complete set of thermodynamic parameters for binding and translocation for the two hydrophobic ion structural analogues, tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB-). A reasonable fit to the data is possible if the dipole potential energy has a magnitude of 5.5 + 0.5 kcal/mol (240 + 20 mV), positive inside, and if the neutral energy contribution for TPP+ and TPB- is -7.0 + 1.0 kcal/mol. These results may also have important implications for small ion interactions with membranes and the energetics of charged groups in membrane proteins.

Animals↗
PubMed1986

Investigation of surface potential asymmetry in phospholipid vesicles by a spin label relaxation method.

In earlier work, Castle and Hubbell (1976) demonstrated the use of a spin-labeled amphiphile as a probe for the electrostatic potential at the outer surface of charged phospholipid vesicles. In recent experiments, we have shown that the hydrophobic anion tetraphenylboron (TPB) promotes transbilayer migration of the probe molecule. Relaxation data recorded following the rapid mixing of the probe with TPB-containing vesicle samples provides information about the electrostatic potentials at both the outer and inner vesicle surfaces. The measured potentials for both surfaces of asymmetrically screened vesicles were found to be in good agreement with theoretical values calculated using their known surface charge density. The method is also sensitive to transmembrane potentials as indicated by the response of the label to potentials created with the use of potassium concentration gradients and valinomycin.

Cyclic N-Oxides↗
PubMed1985

Effects of lipid environment on the light-induced conformational changes of rhodopsin. 1. Absence of metarhodopsin II production in dimyristoylphosphatidylcholine recombinant membranes.

Photolysis of bovine rhodopsin in dimyristoylphosphatidylcholine recombinant membranes results in the production of a relatively stable metarhodopsin I like photointermediate that decays slowly to a species with a broad absorbance maximum centered at about 380 nm [O'Brien, D. F., Costa, L. F., & Ott, R. A. (1977) Biochemistry 16, 1295-1303]. On the basis of the results of a variety of chemical and spectroscopic tests, we show that this process corresponds to the production of free retinal plus opsin and not to the slow production of metarhodopsin II. Electron spin resonance studies using a novel disulfide spin-label that is covalently linked to rhodopsin indicate that the apparent arrest of the protein at the metarhodopsin I stage is not due to simple aggregation of the protein in this short-chain, saturated lipid bilayer but must be understood in terms of the effect of the lipid host on the conformational energies of individual protein molecules. Limited production of metarhodopsin II is observed under acidic conditions. Thus, the rhodopsin-dimyristoylphosphatidylcholine recombinants offer a unique system for the study of the effect of the phospholipid bilayer environment on the conformation of an intrinsic membrane protein.

Animals↗
PubMed1985

Effects of lipid environment on the light-induced conformational changes of rhodopsin. 2. Roles of lipid chain length, unsaturation, and phase state.

When rhodopsin is incorporated into the saturated short-chain phospholipid dimyristoylphosphatidylcholine, photolysis of the protein results in an abnormal sequence of spectral transitions, and the dominant product of metarhodopsin I decay is free retinal plus opsin [Baldwin, P. A., & Hubbell, W. L. (1985) Biochemistry (preceding paper in this issue)]. By incorporation of rhodopsin into a series of phosphatidylcholines of defined composition, we have determined the properties of the lipid environment that are responsible for the altered spectral behavior. Metarhodopsin II is not found in appreciable amounts in bilayers containing acyl chains that are too short (14 or fewer carbon atoms in length), in the presence of only n-alkyl chains, or below the characteristic phase-transition temperature of recombinant membranes. Double bonds are not required for the formation of the metarhodopsin II intermediate, as it is observed in diphytanoylphosphatidylcholine recombinants.

Animals↗
PubMed1984

Stability of "salt bridges" in membrane proteins.

We estimate the free energies of transfer of ionized amino acid side chains in water to both their ion-paired and neutral hydrogen-bonded states in low-dielectric media. The difference between the two free energies corresponds to the proton transfer free energy in a "salt bridge" formed between acidic and basic groups (i.e., lysine and glutamic acid residues). Our approach is to use gas phase proton transfer data, pK values, and experimentally determined solvation energies to estimate the standard state free energy changes involved in transferring amino acid side chains, in both ionized and neutral form, from water (dielectric constant epsilon = 80) to vacuum (epsilon = 1). The familiar expressions for the charging energy of a sphere and dipole are used to interpolate between epsilon = 1 and epsilon = 80. Our results suggest that it costs approximately 10-16 kcal/mol to transfer a salt bridge from water to a medium of epsilon = 2-4, in ionized or neutral form within the resolution of our estimates. The proton transfer energy is thus approximately 0. The tendency of salt bridges to form additional hydrogen bonds in real proteins suggests that the ion pair will be present in most biological systems.

Calorimetry↗
PubMed1983

Electrogenic H+/OH- movement across phospholipid vesicles measured by spin-labeled hydrophobic ions.

Transmembrane pH gradients created across phospholipid vesicles give rise to time-dependent potentials as determined from the EPR spectra of phosphonium ion spin labels in the system. From the time-dependent spectra, the transmembrane H+/OH- current is obtained and hence the current-voltage curve for the vesicle membrane is obtained. The current-voltage curve is linear with a membrane resistance of 3 +/- 2 X 10(9) omega cm2 corresponding to a membrane permeability of 5 +/- 2 X 10(-7) cm/s. This unusually high permeability is further increased by small amounts of lipid oxidation, CHCl3 or the general anesthetic halothane.

Electron Spin Resonance Spectroscopy↗
PubMed1982

Transmembrane electrical currents of spin-labeled hydrophobic ions.

When spin-labeled phosphonium ions are rapidly mixed with phospholipid vesicles, time-dependent changes in the electron paramagnetic resonance spectrum of the spin label are observed. These changes are interpreted in terms of transmembrane transport of the hydrophobic ion, and simple analysis of the data at different membrane potentials is shown to give the binding constant of the ion to both membrane surfaces, the permeability, and current-voltage relationship for the vesicle membrane in the presence of the hydrophobic ion. These results establish the time resolution for methods using the phosphonium ion as a probe of time-dependent potentials across vesicle membranes, as well as provide fundamental information regarding the binding and transport of hydrophobic cations across bilayers. This latter point is significant in view of the fact that hydrophobic cations have not been well characterized in planar bilayers due to their weak binding and low conductance.

Lipid Bilayers↗
PubMed1981

Immunospecific targeting of liposomes to cells: a novel and efficient method for covalent attachment of Fab' fragments via disulfide bonds.

An efficient method for covalently cross-linking 50K Fab' antibody fragments to the surface of lipid vesicles is reported. Coupling up to 600 microgram of Fab'/mumol of phospholipid (about 6000 Fab' molecules per 0.2-micrometer vesicle) is achieved via a disulfide interchange reaction between the thiol group exposed on each Fab' fragment and a pyridyldithio-derivative of phosphatidylethanolamine present in low concentration in the membranes of preformed large unilamellar vesicles. The coupling reaction is efficient, proceeds rapidly under mild conditions, and yields well-defined products. Each vesicle-linked Fab' fragment retains its original antigenic specificity and full capacity to bind antigen. We have used Fab' fragments, coupled to vesicles by this method, to achieve immunospecific targeting of liposomes to cells in vitro. Vesicles bearing antihuman erythrocyte Fab' fragments bind quantitatively to human erythrocytes (at multiplicities up to 5000 0.2-micrometer vesicles per cell) while essentially no binding is observed to sheep or ox red blood cells. Vesicle-cell binding is stable over a pH range from 6 to 8 and is virtually unaffected by the presence of human serum (50%). Cell-bound vesicles retain their aqueous contents and can be eluted intact from cells by treatment with reducing agents (dithiothreitol or mercaptoethanol) at alkaline pH.

Animals↗
PubMed1980

Light-induced interfacial potentials in photoreceptor membranes.

A rapid change in an interfacial electric potential of isolated bovine rod outer segment disk membranes occurs upon illumination. This potential change, which has been detected by the use of spin-labeled hydrophobic ions, apparently occurs within a low dielectric boundary region of the membrane near the external (cytoplasmic) surface and is positive with respect to the aqueous exterior of the disk. The magnitude of the potential change is pH-and temperature-dependent and appears with a first-order half-time of approximately 7 ms at 21 degrees C. A simple model in which one positive charge per bleached rhodopsin is translocated from the cytoplasmic aqueous space into the membrane low dielectric boundary region readily accounts for all experimental observations. The great similarity of the boundary potential change to the R2 phase of the early receptor potential suggests that the two have the same molecular origin.

Animals↗
PubMed1978

Organization of rhodopsin in photoreceptor membranes. 1. Proteolysis of bovine rhodopsin in native membranes and the distribution of sulfhydryl groups in the fragments.

Papain and thermolysin are shown to cleave bovine rhodopsin in native membranes in two temporally distinct steps at room temperature. The final product of the proteolysis consists of two membrane-bound fragments of molecular weights 27 000 (Rh27) and 12 500 (Rh12). The molecular weights are not changed by reduction with dithiothreitol. The two fragments remain closely associated in both the membrane and nondenaturing detergents before and after bleaching and can be selectively cross-linked with carbodiimides. The sulfhydryl chemistry of the cleaved protein in nearly indistinguishable from native rhodopsin, and of the total of six sulfhydryl groups, two are located on Rh12 and four on Rh27. In the membrane-bound protein, two sulfhydryl groups are accessible for modification, one on Rh12 and the other on Rh27. The sulfhydryl on Rh12 is particularly reactive and may be selectively labeled with maleimides. Continuous irradiation with white light induces additional sulfhydryl reactivity on Rh27.

Animals↗