Biomedical subjects

C Weitzman

Publications and source records attributed to C Weitzman.

8 recordsLinked to original sources

PubMed2000

Structure of a glycerol-conducting channel and the basis for its selectivity.

Membrane channel proteins of the aquaporin family are highly selective for permeation of specific small molecules, with absolute exclusion of ions and charged solutes and without dissipation of the electrochemical potential across the cell membrane. We report the crystal structure of the Escherichia coli glycerol facilitator (GlpF) with its primary permeant substrate glycerol at 2.2 angstrom resolution. Glycerol molecules line up in an amphipathic channel in single file. In the narrow selectivity filter of the channel the glycerol alkyl backbone is wedged against a hydrophobic corner, and successive hydroxyl groups form hydrogen bonds with a pair of acceptor, and donor atoms. Two conserved aspartic acid-proline-alanine motifs form a key interface between two gene-duplicated segments that each encode three-and-one-half membrane-spanning helices around the channel. This structure elucidates the mechanism of selective permeability for linear carbohydrates and suggests how ions and water are excluded.

Amino Acid Motifs↗

PubMed1998

In vitro biotinylation provides quantitative recovery of highly purified active lactose permease in a single step.

Consler et al. [Consler, T. G., Persson, B. L., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6934-6938] described a one-step purification of lactose permease, a hydrophobic membrane transport protein, from Escherichia coli. Permease constructs containing a biotin acceptor domain are biotinylated in vivo, followed by solubilization and avidin affinity purification. Although a high degree of purity is obtained, only about 15-20% of the permease is recovered due to incomplete biotinylation. In this communication, a simple modification is described that allows quantitative recovery of highly purified permease. Membranes containing permease with the biotin acceptor domain from the Klebsiella pneumoniae oxaloacetate decarboxylase are extracted with 5 M urea or treated with dicyclohexylcarbodiimide to inactivate F1/Fo ATPase and biotinylated in vitro with biotin ligase, ATP and d-biotin. Subsequently, the membranes are harvested, washed to remove free biotin and solubilized with 2% n-dodecyl-beta-D-maltopyranoside. Biotinylated permease is then purified in one step by affinity chromatography on monomeric avidin-Sepharose. The purified material is homogeneous and exhibits full activity with respect to ligand binding and counterflow.

Anilino Naphthalenesulfonates↗

PubMed1995

Cysteine scanning mutagenesis of helix V in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys (C-less permease), each amino acid residue in putative transmembrane helix V was replaced individually with Cys (from Met145 to Thr163). Of the 19 mutants, 13 are highly functional (60-125% of C-less permease activity), and 4 exhibit lower but significant lactose accumulation (15-45% of C-less permease). Cys replacement of Gly147 or Trp151 essentially inactivates the permease (< 10% of C-less); however, previous studies [Menezes, M. E., Roepe, P. D., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1638; Jung, K., Jung, H., et al. (1995) Biochemistry 34, 1030] demonstrate that neither of these residues is important for activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to C-less permease with the exception of Trp151-->Cys and single Cys154 permeases which are present in reduced amounts. Finally, only three of the single-Cys mutants are inactivated significantly by N-ethylmaleimide (Met145-->Cys, native Cys148, and Gly159-->Cys), and the positions of the three mutants fall on the same face of helix V.

Amino Acid Sequence↗

PubMed1995

Fluorescence of native single-Trp mutants in the lactose permease from Escherichia coli: structural properties and evidence for a substrate-induced conformational change.

Six single-Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87:1638-1642), and fluorescent properties were studied with respect to solvent accessibility, as well as alterations produced by ligand binding. The emission of Trp 33, Trp 78, Trp 171, and Trp 233 is strongly quenched by both acrylamide and iodide, whereas Trp 151 and Trp 10 display a decrease in fluorescence in the presence of acrylamide only and no quenching by iodide. Of the six single-Trp mutants, only Trp 33 exhibits a significant change in fluorescence (ca. 30% enhancement) in the presence of the substrate analog beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). This effect was further characterized by site-directed fluorescent studies with purified single-Cys W33-->C permease labeled with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS). Titration of the change in the fluorescence spectrum reveals a 30% enhancement accompanied with a 5-nm blue shift in the emission maximum, and single exponential behavior with an apparent KD of 71 microM. The effect of substrate binding on the rate of MIANS labeling of single-Cys 33 permease was measured in addition to iodide and acrylamide quenching of the MIANS-labeled protein. Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change in acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J, Frillingos S, Kaback HR, 1995a, Biochemistry 34:8257-8263) that ligand binding induces a conformational change at the C-terminus of helix I such that Pro 28 and Pro 31, which are on one face, become more accessible to solvent, whereas Trp 33, which is on the opposite face, becomes less accessible to the aqueous phase. The findings regarding accessibility to collisional quenchers are also consistent with the predicted topology of the six native Trp residues in the permease.

Acrylamide↗

PubMed1994

Ethanol stimulates the plasma membrane calcium pump from human erythrocytes.

The plasma membrane Ca(2+)-ATPase from human erythrocytes can be stimulated by different treatments such as addition of calmodulin or acidic phospholipids and controlled proteolysis. In this report we show that short chain alkyl alcohols also stimulated this enzyme. At 5% (v/v) ethanol, the maximal velocity of the enzyme was about 2.4-fold higher than in the control, and thus, was also higher than the maximal velocity obtained in the presence of calmodulin (about 2-fold). When ethanol and calmodulin were present simultaneously, the stimulatory effect was additive (3.4-fold stimulation). On the other hand, the stimulatory effect of ethanol was preserved after treatment of the enzyme with trypsin to stimulate the Ca(2+)-ATPase and render it independent of calmodulin, thus suggesting that the interaction of ethanol and calmodulin with the Ca(2+)-ATPase occurred through a different mechanism. Other short chain alkyl alcohols (methanol, n-propanol and n-butanol) stimulated the Ca(2+)-ATPase activity to the same extent than ethanol but with different efficacy. Thus, the larger the carbon number, the lower the concentration needed to get the same maximal stimulation. Ethanol also increased the affinity of the enzyme for ATP to a larger extent and additively, when compared to calmodulin. All the effects of ethanol mentioned above were identically observed on the membrane-bound enzyme (i.e., erythrocyte ghosts) ruling out any effect of the alcohols attributable to the solubilized purified enzyme. Furthermore, Ca2+ transport by inside-out vesicles was also stimulated by ethanol, showing both the same concentration-dependence as the Ca(2+)-ATPase activity and the additive effect observed when calmodulin was also present. The stimulatory effect of ethanol was significant at pharmacological concentrations, thus suggesting potential implications of toxicological relevance.

Adenosine Triphosphate↗

PubMed1994

Fusion proteins as tools for crystallization: the lactose permease from Escherichia coli.

A novel strategy is presented for the crystallization of membrane proteins or other proteins with low solubility and/or stability. The method is illustrated with the lactose permease from Escherichia coli, in which a fusion is constructed between the permease and a 'carrier' protein. The carrier is a soluble, stable protein with its C and N termini close together in space at the surface of the protein, so that the carrier can be introduced into an internal position of the target protein. The carrier is chosen with convenient spectral or enzymatic properties, making the fusion protein easier to handle than the native molecule. Data are presented for the successful construction, expression and purification of a fusion product between lactose permease and cytochrome b(562) from E. coli. The lactose transport activity of the fusion protein is similar to that of wild-type lactose permease, and the fusion product has an absorption spectrum in the visible range which is essentially identical to that of cytochrome b(562). The fusion protein has a higher proportional polar surface area than wild-type permease, and should have better possibilities of forming the strong directional intermolecular contacts required of a crystal lattice.

Journal Article↗

PubMed1994

The lactose permease meets Frankenstein.

The lactose permease (lac) of Escherichia coli is a paradigm for membrane transport proteins. Encoded by the lacY gene, the permease has been solubilized, purified to homogeneity, reconstituted into phospholipid vesicles and shown to catalyse the coupled translocation of beta-galactosides and H+ with a stoichiometry of unity. Circular dichroism and other spectroscopic approaches demonstrate that the purified permease is about 80% helical. Based on hydropathy analysis of the primary amino-acid sequence, a secondary structure has been proposed in which the protein has 12 hydrophobic domains in alpha-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic loops. A variety of other approaches are consistent with the model and demonstrate that both the N and C termini are on the inner surface of the membrane, and studies on an extensive series of lac permease/alkaline phosphatase fusion proteins provide exclusive support for the topological predictions of the 12-helix motif. This presentation concentrates on the use of site-directed fluorescence spectroscopy to study structure-function relationships in the permease.

Amino Acid Sequence↗

PubMed1991

The effect of cholera toxin on human red cell Ca-ATPase.

The effect of cholera toxin on transport Ca-ATPase was studied in membrane fragments from human red cells. A consistently moderate inhibition was found when fragments were previously incubated with toxin in the presence of beta-NAD but not in its absence of after treatment with non-activated toxin. In calmodulin-free preparations, both Ca affinity and maximal rate of hydrolysis were affected whereas only affinity was altered in calmodulin-deficient membranes.

Ca(2+) Mg(2+)-ATPase↗