Small molecule modulation of the Drosophila Slo channel elucidated by cryo-EM.

Slowpoke (Slo) potassium channels display extraordinarily high conductance, are synergistically activated by a positive transmembrane potential and high intracellular Ca concentrations and are important targets for insecticides and antiparasitic drugs. However, it is unknown how these compounds modulate ion translocation and whether there are insect-specific binding pockets. Here, we report structures of Drosophila Slo in the Ca-bound and Ca-free form and in complex with the fungal neurotoxin verruculogen and the anthelmintic drug emodepside.

Non-full-length Water-Soluble CXCR4 and CCR5 Chemokine Receptors: Implication for Overlooked Truncated but Functional Membrane Receptors.

It was posited that functionalities of GPCRs require full-length sequences that are negated by residue deletions. Here we report that significantly truncated nfCCR5 and nfCXCR4 still bind native ligands. Receptor-ligand interactions were discovered from yeast 2-hybrid screening and confirmed by mating selection.

Expression, purification and stabilization of human serotonin transporter from E. coli.

The serotonin transporter belongs to the family of sodium-chloride coupled neurotransmitter transporter and is related to depression in humans. It is therefore an important drug target to support treatment of depression. Recently, structures of human serotonin transporter in complex with inhibitor molecules have been published.

QTY code enables design of detergent-free chemokine receptors that retain ligand-binding activities.

Structure and function studies of membrane proteins, particularly G protein-coupled receptors and multipass transmembrane proteins, require detergents. We have devised a simple tool, the QTY code (glutamine, threonine, and tyrosine), for designing hydrophobic domains to become water soluble without detergents. Here we report using the QTY code to systematically replace the hydrophobic amino acids leucine, valine, isoleucine, and phenylalanine in the seven transmembrane α-helices of CCR5, CXCR4, CCR10, and CXCR7.

Purification of GST-Tagged Proteins.

This protocol describes the purification of recombinant proteins fused to glutathione S-transferase (GST, GST-tagged proteins) by Glutathione Affinity purification. The GST tag frequently increases the solubility of the fused protein of interest and thus enables its purification and subsequent functional characterization. The GST-tagged protein specifically binds to glutathione immobilized to a matrix (e.

Strep-Tagged Protein Purification.

The Strep-tag system can be used to purify recombinant proteins from any expression system. Here, protocols for lysis and affinity purification of Strep-tagged proteins from E. coli, baculovirus-infected insect cells, and transfected mammalian cells are given.

Purification of His-Tagged Proteins.

Ni-NTA affinity purification of His-tagged proteins is a bind-wash-elute procedure that can be performed under native or denaturing conditions. Here, protocols for purification of His-tagged proteins under native, as well as under denaturing conditions, are given. The choice whether to purify the target protein under native or denaturing conditions depends on protein location and solubility, the accessibility of the His tag, and the desired downstream application.

Expression and purification of membrane proteins.

Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways.

Cell-free synthesis of functional and endotoxin-free antibody Fab fragments by translocation into microsomes.

A eukaryotic cell-free system based on Spodoptera frugiperda cells was developed for the convenient synthesis of Fab antibody fragments and other disulfide bridge containing proteins. The system uses (i) a cell lysate that is mildly prepared under slightly reduced conditions, thus maintaining the activity of vesicles derived from the endoplasmic reticulum, (ii) signal peptide dependent translocation into these vesicles, and (iii) a redox potential based on reduced and oxidized glutathione. Monomeric heavy and light immunoglobulin chains are almost completely converted to highly active dimeric Fab joined by intermolecular disulfide bridges without supplementation of chaperones or protein disulfide isomerase.

Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression.

Autologous expression of recombinant human proteins in human cells for biomedical research and product development is often hampered by low expression yields limiting subsequent structural and functional analyses. Following RNA and codon optimization, 50 candidate genes representing five classes of human proteins--transcription factors, ribosomal and polymerase subunits, protein kinases, membrane proteins and immunomodulators--all showed reliable, and 86% even elevated expression. Analysis of three representative examples showed no detrimental effect on protein solubility while unaltered functionality was demonstrated for JNK1, JNK3 and CDC2 using optimized constructs.

Gene optimization mechanisms: a multi-gene study reveals a high success rate of full-length human proteins expressed in Escherichia coli.

The genetic code is universal, but recombinant protein expression in heterologous systems is often hampered by divergent codon usage. Here, we demonstrate that reprogramming by standardized multi-parameter gene optimization software and de novo gene synthesis is a suitable general strategy to improve heterologous protein expression. This study compares expression levels of 94 full-length human wt and sequence-optimized genes coding for pharmaceutically important proteins such as kinases and membrane proteins in E.

Immobilized-metal affinity chromatography (IMAC): a review.

This article reviews the development of immobilized-metal affinity chromatography (IMAC) and describes its most important applications. We provide an overview on the use of IMAC in protein fractionation and proteomics, in protein immobilization and detection, and on some special applications such as purification of immunoglobulins and the Chelex method. The most relevant application-purification of histidine-tagged recombinant proteins-will be reviewed in greater detail with focus of state-of-the-art materials, methods, and protocols, and the limitations of IMAC and recent advances to improve the technology and the methods will be described.

Binding of netrin-4 to laminin short arms regulates basement membrane assembly.

Netrins were first identified as neural guidance molecules, acting through receptors that are members of the DCC and UNC-5 family. All netrins share structural homology to the laminin N-terminal domains and the laminin epidermal growth factor-like domains of laminin short arms. Laminins use these domains to self-assemble into complex networks.

Cleavage and oligomerization of gliomedin, a transmembrane collagen required for node of ranvier formation.

Gliomedin, which has been implicated as a major player in genesis of the nodes of Ranvier, contains two collagenous domains and an olfactomedin-like domain and belongs to the group of type II transmembrane collagens that includes collagens XIII and XVII and ectodysplasin A. One characteristic of this protein family is that constituent proteins can exist in both transmembrane and soluble forms. Recently, gliomedin expressed at the tips of Schwann cell microvilli was found to bind axonal adhesion molecules neurofascin and NrCAM in interactions essential for Na(+)-channel clustering at the nodes of Ranvier in myelinating peripheral nerves.