David H. Singleton

Topology of the pore-region of a K+ channel revealed by the NMR-derived structures of scorpion toxins

The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.

Effect of the activation of phosphatidylinositol 3-kinase by a thiophosphotyrosine peptide on glucose transport in 3T3-L1 adipocytes.

Insulin causes the activation of phosphatidylinositol 3-kinase (PI 3-kinase) through complexation of tyrosine-phosphorylated YMXM motifs on insulin receptor substrate 1 with the Src homology 2 domains of PI 3-kinase. Previous studies with inhibitors have indicated that activation of PI 3-kinase is necessary for the stimulation of glucose transport in adipocytes. Here, we investigate whether this activation is sufficient for this effect. Short peptides containing two tyrosine-phosphorylated or thiophosphorylated YMXM motifs potently activated PI 3-kinase in the cytosol from 3T3-L1 adipocytes. Introduction of the phosphatase-resistant thiophosphorylated peptide into 3T3-L1 adipocytes through permeabilization with Staphylococcus aureus α-toxin stimulated PI 3-kinase as strongly as insulin. However, under the same conditions the peptide increased glucose transport into the permeabilized cells only 20% as well as insulin. Determination of the distribution of the glucose transporter isotype GLUT4 by confocal immunofluorescence showed that GLUT4 translocation to the plasma membrane can account for the effect of the peptide. These results suggest that one or more other insulin-triggered signaling pathways, besides the PI 3-kinase one, participate in the stimulation of glucose transport.

Use of capillary electrophoresis-electrospray ionization mass spectrometry in the analysis of synthetic peptides.

We have constructed a capillary electrophoresis (CE) system with UV detection and have successfully interfaced it to an electrospray ionization mass spectrometry (ES-MS) system. A synthesized fragment of heregulin-beta (212-226) was thought to be a single component by re-injection into an HPLC system, but results from CE-UV-ES-MS indicated that a dehydration product was present in the desired peptide sample. A synthetic heregulin-alpha (177-241) was isolated by preparative HPLC, but re-injection on an analytical system indicated a tailing peak. CE-UV-ES-MS indicated a mixture whose two major components were of the same nominal molecular mass (within experimental error), suggesting the presence of an isomer or a deamidation product. The results show that CE-UV-ES-MS can be used as an orthogonal analytical technique to solve practical problems encountered in peptide synthesis laboratories.

Glucagon-like peptide-1 and analogs: A structure/function analysis

Glucagon-like peptide-1 (GLP-1) is an effective insulin secretagogue with sustained efficacy, with therapeutic potential in treating diabetes [1], As a result there has been much interest is determining its’ receptor bound conformation for rational drug design. In our study, we have identified the important residues for binding by the substitution of amino acids at sites that seemed likely, from an examination of the amino acid sequence and from previously published observations. We have used NMR, using standard methods [2-4], in conjunction with measurements of functional bioactivity to define the receptor-binding structure of GLP-1.

Peptides for elucidating the signal tranduction pathways of CTLA-4 and CD28

CTLA-4, transiently expressed on the surface of activated T-cells, binds to antigen presenting cell ligands B7.1 and B7.2 with 100 fold higher affinity than its competitive receptor, CD28. CD28 promotes T-cell activation upon such binding, however, CTLA-4 bound to B7 ligands potently down regulates cytokine production. Upon binding of B7 to either CTLA-4 or CD28, intracellular signal transduction commences via their cytoplasmic tails. The cascade of proteins binding to these cytoplasmic tails and their signal transduction paths are poorly understood beyond initial recruitment of known signal transduction initiators. Peptides, incorporating a biotin tag, emulating these tails were synthesized as monomeric and dimeric constructs varying in states of phosphorylation. These peptides demonstrate functionality by binding to PI3-kinase and SHP-2 phosphatase, which are known to initially bind to these cytoplasmic tails and thought to be initiators of the signal transduction cascades [1]. Peptide characterizations, as demonstrated by Western blotting analysis, show appropriate functionality.