Erwin London, Ph.D.
Department of Biochemistry and Cell Biology
Center for Structural Biology
Centers for Molecular Medicine
Stony Brook University
Stony Brook, NY 11794-5215
Office telephone: 631-632-8564
Membrane Protein Structure: Determining the Rules for Membrane Protein Translocation and Folding.
Our group is studying membrane protein structure and function by combining spectroscopic methods, such as fluorescence, with chemical, biochemical, immunochemical and molecular biological approaches. We are interested in the determining membrane protein structure and the origin of specific lipid-protein and protein-protein interactions. At present, we are concentrating on protein toxins that penetrate and translocate across cell membranes, such as diphtheria toxin. Our aim is to understand the mechanism of membrane penetration and translocation by this toxin. This should have important implications for protein translocation in general, as well as the design of therapeutic agents and vaccines for bacterial infections. To analyze the structure of diphtheria toxin in membrane site-directed mutagenesis is used to introduce site-directed fluorescence labels. The location of the residues relative to the membrane is then determined using antibody binding or a fluorescence quenching technique (parallax analysis) developed in our lab (see below). In this method the difference in the amount of fluorescence quenching of the fluorescently-labeled residue by lipids carrying quenching groups at different positions (depths) is used to calculate the depth of the fluorescent group.
We are also using studying the relationship between amino acid sequence and structure using simple transmembrane helices. Such helices are the main structural element within membrane proteins. We have begun studying the effect of introducing hydrophilic residues into a hydrophobic sequence. The identity, number and position of these residues within the sequence are being varied. Their structure and location within the bilayer is analyzed using fluorescence, fluorescence quenching, circular dichroism and other spectroscopic techniques. This will allow us to derive basic rules for membrane protein folding.
The Structure and Function of Cholesterol-Rich Membrane Domains
Together with Dr. Deborah Brown in the Dept. Of Biochemistry and Cell Biology we have been studying the structure and function of lipid domains enriched in cholesterol and sphingolipid. These domains have been proposed to have a functional role in processes such as viral and toxin entry into cells, protein sorting among organelles, signal transduction, prion formation and amyloid formation. Our studies involve determining the basic principles that drive the formation of these domains and regulate their lipid and protein composition.
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- Asuncion-Punzalan, E., and London, E. (1995) "Control of the Depth of Molecules within Membranes by Polar Groups: Determination of the Location of Anthracene Labeled Probes in Model Membranes by Parallax Analysis of Nitroxide-Labeled Phospholipid Induced Fluorescence Quenching" Biochemistry 34, 11460-11466.
- Tortorella, D., Sesardic, D., Dawes, C.S., and London, E. (1995) "Immunochemical Analysis of the Structure of Diphtheria Toxin Shows All Three Domains Undergo Structural Changes at Low pH" J. Biol. Chem. 270, 27439-27445.
- Tortorella, D., Sesardic, D., Dawes, C.S., and London, E. (1995) "Immunochemical Analysis of the Structure of Diphtheria Toxin Shows All Three Domains Penetrate Across Model Membranes" J. Biol. Chem. 270, 27446-27452.
- Kachel, K., Asuncion-Punzalan, E., and London, E. (1995) "Anchoring of Trp and Tyr Analogs at the Hydrocarbon-Polar Boundary in Membranes: Parallax Analysis of Fluorescence Quenching Induced by Nitroxide-Labeled Phospholipids" Biochemistry 34, 15475-15479.
- Paliwal, R., and London, E. (1996) "Comparison of the Conformation, Hydrophobicity and Model Membrane Interaction of Diphtheria Toxin to That of Formaldehyde-Treated Toxin (Diphtheria Toxoid): Formaldehyde Stabilization of the Native Conformation Inhibits Changes That Allow Membrane Insertion", Biochemistry 35, 2374-2379.
- Ren, J., Lew, S., Wang, Z., and London, E. (1997) "Transmembrane Orientation of Hydrophobic -Helices is Regulated by the Relationship of Helix Length to Bilayer Thickness and by Cholesterol Concentration", Biochemistry 36, 10213-10220.
- Ahmed, S.N., Brown, D.A., and London, E. (1997) "On the Origin of Sphingolipid/Cholesterol Rich Detergent-Insoluble Domains in Cell Membranes: Physiological Concentrations of Cholesterol and Sphingolipid Induce Formation of a Detergent-Insoluble Liquid Ordered Phase in Model Membranes", Biochemistry 36, 10944-10953.
- Wang, Y., Malenbaum, S.E., Kachel, K., Zhan, H., Collier, R.J., and London, E. (1997) "Identification of Shallow and Deep Membrane Penetrating Conformations of Diphtheria Toxin T Domain That Are Regulated by T Domain Concentration and Bilayer Width", J. Biol. Chem. 272, 25091-25098.
- Wang, Y., Kachel, K., Pablo, L., and London, E. (1997) "Use of Trp Mutations to Evaluate the Conformational Behavior and Membrane-Insertion of A and B Chains in Whole Diphtheria Toxin", Biochemistry 36, 13600-13608.
- Schroeder, R., Ahmed, S.N., Zhu, Y., London, E., and Brown, D.A. (1998) "How Cholesterol and Sphingolipid Enhance the Triton X-100-Insolubility of GPI-Anchored Proteins by Promoting Formation of Detergent-Insoluble Ordered Membrane Domains", J. Biol. Chem. 273, 1150-1157.
- Asuncion-Punzalan, E., Kachel, K., and London, E. (1998) "Polar Molecules Can Locate at Both Shallow and Deep Locations in Membranes: The Behavior of Dansyl and Related Probes", Biochemistry 37, 4603-4611.
- Kaiser, R.D., and London, E. (1998) "Location of Diphenylhexatriene (DPH) Derivatives Within Membranes: Comparison of Different Fluorescence Quenching Analyses of Membrane Depth", Biochemistry 37, 8180-8190. (Review) Brown, D.A., and London, E. (1998) "Origin and Structure of Ordered Lipid Domains in Biological Membranes" J. Memb. Bio. 164, 103-114.
- Brown, D.A., and London, E. (1998) "Functions of Lipid Rafts in Biological Membranes", Ann. Rev. Cell Dev. Bio. 14, 111-136.
- Kachel, K., Asuncion-Punzalan, E., and London, E. (1998) "The Location of Molecules with Charged Groups in Membranes", Biochim. Biophys. Acta 1374, 63-76.
- Kachel, K., Ren, J., Collier, R.J., and London, E. (1998) "Identifying Transmembrane States and Defining the Membrane Insertion Boundaries of Hydrophobic Helices: The Conformation of TH8 and TH9 in Membrane-Inserted Diphtheria Toxin T Domain", J. Biol. Chem. 273, 22950-22956.
- Malenbaum, S.E., Collier, R.J., and London, E. (1998) "Membrane Insertion of Helices TH1, TH5 and TH9 of the T Domain of Diphtheria Toxin Probed With Single Trp Mutants", Biochemistry 37, 17915-17922.
- Ren, J., Collier, R.J., and London, E. (1999) "Role of Ionizable Residues at the Tips of Hydrophobic Helices in the Transmembrane Insertion of the T Domain of Diphtheria Toxin", Biochemistry 38, 976-984.
- Ren, J., Lew, S., Wang, J., and London, E. (1999) "Control of Transmembrane Orientation and Interhelical Interactions within Membranes by Hydrophobic Helix Length", Biochemistry 38, 5905-5912.
- Ren, J., Kachel, K., Malenbaum, S.E., Collier, R.J. and London, E. (1999) "Interaction of Diphtheria Toxin T Domain with Molten Globule Like Proteins and Its Implications for Translocation", Science 284, 955-957.
- Sharpe, J.C., and London, E. (1999) "Diphtheria Toxin Forms Pores of Different Sizes Depending On Its Concentration in Membranes: Probable Relationship to Oligomerization", J. Memb. Biol. 171, 209-221.
- Sharpe, J.C., Kachel, K., and London, E. (1999), The Effects of Inhibitors Upon Pore Formation by Diphtheria Toxin and Diphtheria Toxin T Domain", J. Memb. Biol. 171, 223-233.
- Xu, X., and London, E. (2000) "The Effect of Sterol Structure on Membrane Lipid Domains Reveals How Cholesterol Can Induce Lipid Domain Formation", Biochemistry 39, 844-849. 70. Xu, X., and London, E. (2000) "The Effect of Sterol Structure on Membrane Lipid Domains Reveals How Cholesterol Can Induce Lipid Domain Formation" Biochemistry 39, 844-849.
- Brown, D.A., and London, E. (2000) "Structure and Function of Sphingolipid- and Cholesterol-rich Rafts" J. Biol. Chem. 275, 17221-17224.
- Lew, S., and London, E. (2000) "The Effect of Polar/Ionizable Residues Within the Core of Hydrophobic Helices on Their Behavior Within Lipid Bilayers" Biochemistry39, 9632-9640.
- London, E., Brown, D.A., and Xu, X. (2000) "Fluorescence Quenching Assay of Sphingolipid/Phospholipid Phase Separation" Meth. Enzymol. 312, 272-290.
- London, E., and Brown, D.A. (2000) "Insolubility of Lipids in Triton X-100: Physical Origin and Relationship to Sphingolipid/Cholesterol Membrane Domains (rafts)" Biochim. Biophys. Acta 1508, 182-195. Xu, X., Bittman, R., Duportail, G.,
- Heissler, D., Vilcheze, C., and London, E. (2001) "Effect of the Structure of Natural Sterols and Sphingolipids on the Formation of Ordered Sphingolipid/Sterol Domains (Rafts): Comparison of Cholesterol to Plant, Fungal and Disease-Associated Sterols, and Comparison of Sphingomyelin, Cerebrosides and Ceramide" J. Biol. Chem. 276, 33540-33546.
- London, E., and Ladokhin, A.S. (2002) "Measuring the Depth of Amino Acid Residues in Membrane-Inserted Peptides by Fluorescence Quenching" in Current Topics in Membranes: Peptide-Lipid Interactions (Simon, S.A., and McIntosh, T.J. Eds.) Vol. 52 Academic Press, 89-115.
- Hammond, K., Caputo, G.A., and London, E. (2002) "Interaction of the Membrane-Inserted Diphtheria Toxin T Domain with Peptides and Its Possible Implications for Chaperone-like T Domain Behavior" Biochemistry 41, 3243-3253.
- Rosconi, M.P., and London, E. (2002) "Topography of Helices 5-7 in Membrane- Inserted Diphtheria Toxin T Domain: Identification and Insertion Boundaries of Two Hydrophobic Sequences That Do Not Form a Stable Transmembrane Hairpin" J. Biol.Chem., 277, 16517-16527.
- London, E. (2002) "Insights into Lipid Domain/Raft Structure and Formation from Experiments in Model Membranes" Curr. Opin. Struct. Bio., 12, 480-486.
- Caputo, G.A., and London, E. (2003) "Using a Novel Dual Fluorescence Quenching Assay for Measurement of Trp Depth Within Lipid Bilayers to Determine Hydrophobic Alpha-Helix Locations Within Membranes" Biochemistry, in press
- Caputo, G.A., and London, E. (2003) "Cumulative Effects of Amino Acid Substitutions and Hydrophobic Mismatch Upon the Transmembrane Stability and Conformation of Hydrophobic Alpha-Helices" Biochemistry, in press
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