Preparation and Characterization of the Extracellular Domain of Human Sid-1

In C. elegans, the cell surface protein Sid-1 imports extracellular dsRNA into the cytosol of most non-neuronal cells, enabling systemic spread of RNA interference (RNAi) throughout the worm. Sid-1 homologs are found in many other animals, although for most a function for the protein has not yet been established. Sid-1 proteins are composed of an N-terminal extracellular domain (ECD) followed by 9–12 predicted transmembrane regions. We developed a baculovirus system to express and purify the ECD of the human Sid-1 protein SidT1. Recombinant SidT1 ECD is glycosylated and spontaneously assembles into a stable and discrete tetrameric structure. Electron microscopy (EM) and small angle x-ray scattering (SAXS) studies reveal that the SidT1 ECD tetramer is a compact, puck-shaped globular particle, which we hypothesize may control access of dsRNA to the transmembrane pore. These characterizations provide inroads towards understanding the mechanism of this unique RNA transport system from structural prospective

Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae

The SLC13 transporter family, whose members play key physiological roles in the regulation of fatty acid synthesis, adiposity, insulin resistance, and other processes, catalyzes the transport of Krebs cycle intermediates and sulfate across the plasma membrane of mammalian cells. SLC13 transporters are part of the divalent anion:Na(+) symporter (DASS) family that includes several well-characterized bacterial members. Despite sharing significant sequence similarity, the functional characteristics of DASS family members differ with regard to their substrate and coupling ion dependence. The publication of a high resolution structure of dimer VcINDY, a bacterial DASS family member, provides crucial structural insight into this transporter family. However, marrying this structural insight to the current functional understanding of this family also demands a comprehensive analysis of the transporter's functional properties. To this end, we purified VcINDY, reconstituted it into liposomes, and determined its basic functional characteristics. Our data demonstrate that VcINDY is a high affinity, Na(+)-dependent transporter with a preference for C4- and C5-dicarboxylates. Transport of the model substrate, succinate, is highly pH dependent, consistent with VcINDY strongly preferring the substrate's dianionic form. VcINDY transport is electrogenic with succinate coupled to the transport of three or more Na(+) ions. In contrast to succinate, citrate, bound in the VcINDY crystal structure (in an inward-facing conformation), seems to interact only weakly with the transporter in vitro. These transport properties together provide a functional framework for future experimental and computational examinations of the VcINDY transport mechanism

Microbial Glycoside Hydrolases for Biomass Utilization in Biofuels Applications

Renewable biomass is predicted to have the potential to meet at least a quarter of the world demand for transportation fuel, but to do so both terrestrial lignocellulosic as well as marine algal resources need to be efficiently utilized. In the processes where these biomasses are converted into different types of energy carriers (for example fuel-alcohols e.g. ethanol or butanol) microbial glycoside hydrolases (GHs) have a role in the saccharification process. During saccharification polymeric carbohydrate resources (e.g. starch, cellulose or hemicellulose) are hydrolyzed into mono and oligosaccharides that can be utilized by the organism selected to ferment these carbohydrates into the desired energy-carrier. This chapter aims to shed light on different processing alternatives for the conversion of lignocellulose or algal starch into mono or oligosaccharides, and what roles the microbial GHs have as processing aids in these conversions