Expression, purification, and contaminant detection for structural studies of Ralstonia metallidurance ClC protein rm1.

Single-particle electron cryo-microscopy (cryo-EM) has become a popular method for high-resolution study of the structural and functional properties of proteins. However, sufficient expression and purification of membrane proteins holds many challenges. We describe methods to overcome these obstacles using ClC-rm1, a prokaryotic chloride channel (ClC) family protein from Ralstonia metallidurans, overexpressed in Escherichia coli (E.

Biochemical and biophysical approaches to study the structure and function of the chloride channel (ClC) family of proteins.

The chloride channel (ClC) protein family comprises both chloride (Cl(-)) channels and chloride/proton (Cl(-)/H(+)) antiporters. In prokaryotes and eukaryotes, these proteins mediate the movement of Cl(-) ions across the membrane. In eukaryotes, ClC proteins play a role in the stabilization of membrane potential, epithelial ion transport, hippocampal neuroprotection, cardiac pacemaker activity and vesicular acidification.

Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome.

Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics.

Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins.

Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins.

Cryo-electron microscopy of membrane proteins.

Electron crystallography is used to study membrane proteins in the form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.

Merging of image data in electron crystallography.

Electron crystallography of membrane proteins uses cryo-transmission electron microscopy to record images and diffraction patterns of frozen-hydrated 2D crystals. Each two-dimensional (2D) crystal is only imaged once, at one specific tilt angle, and the recorded images can be automatically processed with the 2dx/MRC software package. Processed image data from non-tilted and tilted 2D crystals then need to be merged into a 3D reconstruction of the membrane protein structure.

Preparation of 2D crystals of membrane proteins for high-resolution electron crystallography data collection.

Electron crystallography is a powerful technique for the structure determination of membrane proteins as well as soluble proteins. Sample preparation for 2D membrane protein crystals is a crucial step, as proteins have to be prepared for electron microscopy at close to native conditions. In this review, we discuss the factors of sample preparation that are key to elucidating the atomic structure of membrane proteins using electron crystallography.

Huntingtin facilitates polycomb repressive complex 2.

Huntington's disease (HD) is caused by expansion of the polymorphic polyglutamine segment in the huntingtin protein. Full-length huntingtin is thought to be a predominant HEAT repeat alpha-solenoid, implying a role as a facilitator of macromolecular complexes. Here we have investigated huntingtin's domain structure and potential intersection with epigenetic silencer polycomb repressive complex 2 (PRC2), suggested by shared embryonic deficiency phenotypes.

Oligomeric structure and functional characterization of the urea transporter from Actinobacillus pleuropneumoniae.

Urea transporters (UTs) facilitate urea permeation across cell membranes in prokaryotes and eukaryotes. Bacteria use urea as a means to survive in acidic environments and/or as a nitrogen source. The UT from Actinobacillus pleuropneumoniae, ApUT, the pathogen that causes porcine pleurisy and pneumonia, was expressed in Escherichia coli and purified.

The Affinity Grid: a pre-fabricated EM grid for monolayer purification.

We have recently developed monolayer purification as a rapid and convenient technique to produce specimens of His-tagged proteins or macromolecular complexes for single-particle electron microscopy (EM) without biochemical purification. Here, we introduce the Affinity Grid, a pre-fabricated EM grid featuring a dried lipid monolayer that contains Ni-NTA lipids (lipids functionalized with a nickel-nitrilotriacetic acid group). The Affinity Grid, which can be stored for several months under ambient conditions, further simplifies and extends the use of monolayer purification.

Towards automated screening of two-dimensional crystals.

Screening trials to determine the presence of two-dimensional (2D) protein crystals suitable for three-dimensional structure determination using electron crystallography is a very labor-intensive process. Methods compatible with fully automated screening have been developed for the process of crystal production by dialysis and for producing negatively stained grids of the resulting trials. Further automation via robotic handling of the EM grids, and semi-automated transmission electron microscopic imaging and evaluation of the trial grids is also possible.

WaaL of Pseudomonas aeruginosa utilizes ATP in in vitro ligation of O antigen onto lipid A-core.

waaL has been implicated as the gene that encodes the O-antigen ligase. To date, in vitro biochemical evidence to prove that WaaL possesses ligase activity has been lacking due to the difficulty of purifying WaaL and unavailability of substrates. Here we describe the purification of WaaL, a membrane protein with 11 potential transmembrane segments from Pseudomonas aeruginosa, and the development of an in vitro O-antigen ligase assay.

Biochemical characterization of MsbA from Pseudomonas aeruginosa.

Lipopolysaccharide of Pseudomonas aeruginosa is a major constituent of the outer membrane, and it is composed of three distinct regions: lipid A, core oligosaccharide, and O antigen. Lipid A and core oligosaccharides (OS) are synthesized and assembled at the cytoplasmic side of the inner membrane and then translocated to the periplasmic side of the membrane where lipid A-core becomes the acceptor of the O antigens. Here we show that MsbA encoded by pA4997 of the P.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes.

Coexistence of two distinct versions of O-antigen polymerase, Wzy-alpha and Wzy-beta, in Pseudomonas aeruginosa serogroup O2 and their contributions to cell surface diversity.

Assembly of B-band lipopolysaccharide (LPS) in Pseudomonas aeruginosa follows a Wzy-dependent pathway, requiring the O-antigen polymerase Wzy and other proteins. The peptide sequences of the wzy(alpha) product from strains of serotypes O2, O5, and O16 are identical, but the O units in O5 are alpha-glycosidically linked, while those in O2 and O16 are beta-linked. We hypothesized that a derivative of the D3 bacteriophage wzy(beta) is present in the chromosomes of O2 and O16 and that this gene is responsible for the beta-linkage.

Parallels in rRNA processing: conserved features in the processing of the internal transcribed spacer 1 in the pre-rRNA from Schizosaccharomyces pombe.

Despite the large differences in their length and nucleotide composition, comparative analyses of the internal transcribed spacer 1 (ITS1) of widely divergent eukaryotes have suggested a simple core structure consisting of a central extended hairpin and lesser hairpin structures at the maturing junctions [Lalev, A. I., and Nazar, R.

Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide.

The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P.

A RAC protein-binding site in the internal transcribed spacer 2 of Pre-rRNA transcripts from Schizosaccharomyces pombe.

The interdependence of steps in the processing of the eukaryotic preribosomal rRNA transcripts indicate that rRNA processing, at least in part, acts as a quality control mechanism to help ensure that only functional rRNA is incorporated into mature ribosomes. In search of structural components that underlie this interdependence, we have isolated a large protein complex or RAC that contains an independent binding site for all four of the transcribed spacers in the nascent pre-rRNA. In this study the RAC-binding site in the internal transcribed spacer 2 sequence of Schizosaccharomyces pombe rRNA transcripts was identified, and the influence of this site on rRNA maturation was assessed.