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W L Hubbell

Publications and source records attributed to W L Hubbell.

12 recordsLinked to original sources
PubMed1992

Molecular characterization of helix-loop-helix peptides.

A class of regulators of eukaryotic gene expression contains a conserved amino acid sequence responsible for protein oligomerization and binding to DNA. This structure consists of an arginine- and lysine-rich basic region followed by a helix-loop-helix motif, which together mediate specific binding to DNA. Peptides were prepared that span this motif in the MyoD protein; in solution, they formed alpha-helical dimers and tetramers. They bound to DNA as dimers and their alpha-helical content increased on binding. Parallel and antiparallel four-helix models of the DNA-bound dimer were constructed. Peptides containing disulfide bonds were engineered to test the correctness of the two models. A disulfide that is compatible with the parallel model promotes specific interaction with DNA, whereas a disulfide compatible with the antiparallel model abolishes specific binding. Electron paramagnetic resonance (EPR) measurements of nitroxide-labeled peptides provided intersubunit distance measurements that also supported the parallel model.

Amino Acid Sequence↗
PubMed1992

Determination of electrostatic potentials at biological interfaces using electron-electron double resonance.

A new general method for the determination of electrostatic potentials at biological surfaces is presented. The approach is based on measurement of the collision frequency of a charged nitroxide in solution with a nitroxide fixed to the surface at the point of interest. The collision frequency is determined with 14N:15N double label electron-electron double resonance (ELDOR). As a test, the method is shown to give values for phospholipid bilayer surface potentials consistent with the Gouy-Chapman theory, a simple model shown by many independent tests to accurately describe charged, planar surfaces. In addition, the method is applied to determine the electrostatic potential near the surface of DNA. The results indicate that the potential is significantly smaller than that predicted from Poisson-Boltzmann analysis, but is in qualitative agreement with that predicted by Manning's theory of counter ion condensation. The method is readily extended to measurement of surface potentials of proteins.

Aminacrine↗
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↗
PubMed1978

Organization of rhodopsin in photoreceptor membranes. 2. Transmembrane organization of bovine rhodopsin: evidence from proteolysis and lactoperoxidase-catalyzed iodination of native and reconstituted membranes.

Proteolysis of reconstituted membranes with papain and thermolysin reveals the existence of two rhodopsin populations: one susceptible to proteolysis and the other protected. The susceptible population corresponds to rhodopsin molecules with the same orientation as rhodopsin in the native membrane, while the protected population corresponds to "inverted" rhodopsin molecules only found in reconstituted membranes. Using an iodination enhancement probe, we demonstrate that lactoperoxidase catalyzes iodination of rhodopsin exclusively on the external surface of these sealed reconstituted vesicles. Furthermore, we find that both rhodopsin populations in reconstituted membranes (normal and inverted) are readily iodinated by lactoperoxidase, providing definitive evidence that the rhodopsin polypeptide spans the membrane thickness. Additional conclusions from these experiments are discussed in terms of a model for the folding of the rhodopsin polypeptide in the membrane.

Animals↗
PubMed1978

Estimation of transmembrane pH gradients from phase equilibria of spin-labeled amines.

Spin-labeled secondary amines have been used to measure transmembrane proton gradients in sonicated liposomes. The electron paramagnetic resonance spectra of these probes show changes in the ratio of membrane associated to free aqueous probe as a function of transmembrane pH gradient. As the pH gradient is increased, inside acidic, the amount of membrane associated probe increases. The results are accounted for by a simple thermodynamic theory.

Amines↗
PubMed1978

Estimation of transmembrane potentials from phase equilibria of hydrophobic paramagnetic ions.

Positively charged hydrophobic spin labels have been synthesized which respond to transmembrane potentials in sonicated liposomes. Electron paramagnetic resonance spectroscopy is used to show that the distribution of these probes between aqueous and membrane phases changes as a function of transmembrane potential. When liposomes are made more inside-negative, the fraction of membrane associated probe increases while the fraction of probe in the aqueous phase decreases. The results are in quantitative agreement with a simple equilibrium thermodynamic theory which allows estimation of absolute transmembrane potentials in phospholipid vesicles.

Electron Spin Resonance Spectroscopy↗
PubMed1978

Stability of rhodopsin in detergent solutions.

The thermal stability of lipid-free rhodopsin in solutions of a homologous series of alkyltrimethylammonium bromide detergents and one nonionic detergent, dodecyl-beta-maltoside, has been studied as a function of detergent concentration. Rhodopsin thermal stability increases with increasing chain length within the homologous series of ionic detergents, and for chain lengths greater than 10 carbon atoms increases with increasing detergent concentration up to a "critical" concentration that depends on the chain length. Stability also increases with increasing detergent concentration for rhodopsin in solutions of the nonionic detergent. These results may be rationalized in terms of the dependence of micelle packing density on the detergent chain length, head group, and concentration.

Animals↗
PubMed1978

Reactions of the sulfhydryl groups of membrane-bound bovine rhodopsin.

Reactions of the sulfhydryl groups of bovine rhodopsin in rod outer segment membranes have been investigated using 4,4'-dithiopyridine. This reagent is uncharged at neutral pH and rapidly equilibrates across phospholipid bilayers. Membrane-bound rhodopsin has two kinetically distinguishable sulfhydryl groups reactive to the reagent, this stoichiometry being unchanged by bleaching provided the sulfhydryl reactions themselves are carried out in the dark. The rates of the reactions, however, are substantially increased by bleaching. Irradiation of bleached membranes, either with white light or wavelengths in the neighborhood of 475 nm, results in an increase in the number of reactive sulfhydryls relative to that found for bleached membranes in the dark. A component of the light-driven reaction is dependent on the Ca2+ content of the medium.

Animals↗
PubMed1976

Estimation of membrane surface potential and charge density from the phase equilibrium of a paramagnetic amphiphile.

The distribution of a paramagnetic amphiphile, N,N-dimethyl-N-nonyl-N-tempoylammonium ion, between the membranes of charged phospholipid vesicles and the surrounding aqueous medium was studied by electron paramagnetic resonance spectroscopy. By systematically varying the surface charge of the vesicles and the aqueous electrolyte concentration, the distribution was shown to indicate vesicle surface potential. At each fixed phospholipid composition, the surface potential exhibited a dependence on aqueous NaCl concentration very similar to that predicted by the Gouy equation. The ability to sense and quantitate surface potentials makes this facile and sensitive technique of value in the study of cell and organelle surfaces.

Electron Spin Resonance Spectroscopy↗
PubMed1976

Localization of molecular halothane in phospholipid bilayer model nerve membranes.

The molecular motion and distribution of the inhalation anesthetic halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in a phospholipid bilayer model nerve membrane preparation was studied using fluorine nuclear magnetic resonance. Bilayers containing stable free radicals at known depths were studied to measure possible localization of halothane within certain areas of the bilayer. Bilayer suspensions containing manganese ions in the aqueous phase were used to test the partition of halothane between the aqueous and lipid phases. It was found that halothane rapidly achieves complete exchange throughout the bilayer and the surrounding aqueous phase. The results provide experimental evidence against the formation of anesthetic clathrates hypothesized by Pauling and Miller in their theories of anesthesia.

Fluorides↗