Andrew V. Kralicek

Replication Termination in Escherichia coli: Structure and Antihelicase Activity of the Tus-Ter Complex

SUMMARY The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process.

Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants

Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase. During processing of affected tubers, the high temperatures involved in baking and frying cause the Maillard reaction between reducing sugars and free amino acids, resulting in the accumulation of acrylamide. cDNA clones with deduced proteins homologous to known invertase inhibitors were isolated and the two most abundant forms, termed INH1 and INH2, were shown to possess apoplastic and vacuolar localization, respectively. The INH2 gene showed developmentally regulated alternative splicing, so, in addition to the INH2α transcript encoding the full-length protein, two hybrid mRNAs (INH2β*A and INH2β*B) that encoded deduced vacuolar invertase inhibitors with divergent C-termini were detected, the result of mRNA splicing of an upstream region of INH2 to a downstream region of INH1. Hybrid RNAs are common in animals, where they may add to the diversity of the proteome, but are rarely described in plants. During cold storage, INH2α and the hybrid INH2β mRNAs accumulated to higher abundance in cultivars resistant to cold-induced sweetening than in susceptible cultivars. Increased amounts of invertase inhibitor may contribute to the suppression of acid invertase activity and prevent cleavage of sucrose. Evidence for increased RNA splicing activity was detected in several resistant lines, a mechanism that in some circumstances may generate a range of proteins with additional functional capacity to aid adaptability.

Insights into subunit interactions within the insect olfactory receptor complex using FRET

Insect olfactory receptors (ORs) are a novel family of ligand-gated cation channels that can respond to volatile organic compounds at low concentrations. They are involved in the detection of odorants associated with mate recognition, food localisation and predator avoidance. These receptors form a complex that is currently thought to contain at least two subunit members: the non-canonical Orco ion channel subunit and a ligand-binding receptor subunit. The integral membrane proteins SNMP1 and 2 are also associated with olfactory function, with SNMP1 required for cis-vaccinyl acetate reception in Drosophila melanogaster. In order to investigate protein-protein interactions among these membrane proteins we measured intermolecular Förster/Fluorescence Resonance Energy Transfer (FRET) in live insect cells by acceptor photobleaching. Fusion proteins containing Cyan Fluorescent Protein or Yellow Fluorescent Protein were produced using baculovirus-mediated expression in High Five™ cells. The majority of the recombinant products were of the expected size for the fusion proteins and located within intracellular membranes. We were able to show FRET efficiencies providing evidence for homomeric and heteromeric interactions of the ligand-binding OR, Or22a, and Orco (Or22a-Or22a, Or22a-Orco, Orco-Orco). There was no evidence for an interaction between SNMP1 and Orco or between SNMP2 and Orco or Or22a. However, fusion proteins of SNMP1 and Or22a did show an interaction by FRET, suggesting SNMP1 may interact with at least some insect olfactory receptor complexes. In summary, this study supports previously observed homomeric and heteromeric interactions between Orco and the ligand-binding OR, Or22a, and identifies a novel interaction between Or22a and SNMP1.

Recombinant expression, detergent solubilisation and purification of insect odorant receptor subunits

Insect odorant receptors (ORs) are seven transmembrane domain proteins that comprise a novel family of ligand-gated non-selective cation channels. The functional channel is made up of an odour activated ligand-binding OR and the OR co-receptor, Orco. However, the structure, stoichiometry and mechanism of activation of the receptor complex are not well understood. Here we demonstrate that baculovirus-mediated Sf9 cell expression and wheat germ cell-free expression, but not Escherichia coli cell-based or cell-free expression, can be used successfully to over-express a selection of insect ORs. From a panel of 19 detergents, 1%w/v Zwittergent 3-16 was able to solubilise five Drosophila melanogaster ORs produced from both eukaryotic expression systems. A large-scale purification protocol was then developed for DmOrco and the ligand-binding receptor, DmOr22a. The proteins were nickel-affinity purified using a deca-histidine tag in a buffer containing 0.2 mM Zwittergent 3-16, followed by size exclusion chromatography. These purified ORs appear to form similarly sized protein-detergent complexes when isolated from both expression systems. Circular dichroism analysis of both purified proteins suggests they are folded correctly. We also provide evidence that when DmOrco is expressed in Sf9 cells it undergoes post translational modification, probably glycosylation. Finally we show that the recombinant ORs can be incorporated into pre-formed liposomes. The ability to recombinantly express and purify insect ORs to homogeneity on a preparative scale, as well as insert them into liposomes, is a major step forward in enabling future structural and functional studies, as well as their use in OR based biosensors.

Cell-free synthesis and combinatorial selective 15N-labeling of the cytotoxic protein amoebapore A from Entamoeba histolytica

Amoebapore A is a pore-forming protein produced by the pathogenic parasite Entamoeba histolytica, which causes human amoebic dysentery. The pore-forming activity of amoebapore A is regulated by pH-dependent dimerization, a prerequisite for membrane insertion and pore formation. Understanding of these important processes has been hampered by the cytotoxicity of amoebapore A, which prevents the production of this protein in cell-based expression systems. In this study, a protocol for the cell-free production of active recombinant amoebapore A is presented. Protein yields of approximately 500 microg/ml of cell-free reaction were achieved. Recombinant amoebapore A was purified using a three-step procedure. To facilitate the structural characterization of the dimeric and pore forms, we adapted the cell-free system to isotope label amoebapore A for NMR studies. The preliminary assignment of a 2D 1H-15N HSQC spectrum of a uniformly 13C/15N-labeled sample was achieved using a combinatorial selective 15N-labeling approach coupled with available 1H(N) chemical shift data, resulting in the unambiguous assignment of resonances from 55 of the 77 residues. To confirm these results and obtain the full sequence-specific assignments of the 2D 1H-15N HSQC spectrum, a 3D HNCA spectrum was recorded. These assignment data will be used to aid the characterization of amoebapore A dimer formation and membrane insertion.

A Cell-Free Expression Screen to Identify Fusion Tags for Improved Protein Expression

Cell-free protein synthesis can now be routinely used for the rapid screening of protein expression at the microliter level using PCR-amplified templates. However, identification of the optimal expression construct for a target protein can still be a problem. A rapid cell-free procedure is described here for the systematic assessment of a range of diverse fusion tags on the expression and solubility of any given target protein. Overlap/extension PCR is used to fuse a library of T7 promoter (T7p)-tag fragments with a gene-T7terminator (T7ter) fragment to produce cell-free expression templates encoding different fusion proteins. These constructs are then expressed in a series of small-scale (50 μL) Escherichia coli cell-free reactions and SDS-PAGE analysis is used to identify the optimal fusion tag(s). This screen is particularly useful for the identification of expression constructs for proteins that are normally poorly expressed or are insoluble.

Functional implications of large backbone amplitude motions of the glycoprotein 130-binding epitope of interleukin-6.

Human interleukin (IL)‐6 plays a pivotal role in the immune response, hematopoiesis, the acute‐phase response, and inflammation. IL‐6 has three distinct receptor epitopes, termed sites I, II, and III, that facilitate the formation of a signaling complex. IL‐6 signals via a homodimer of glycoprotein 130 (gp130) after initially forming a heterodimer with the nonsignaling α‐receptor [IL‐6 α‐receptor (IL‐6R)] via site I. Here, we present the backbone dynamics of apo‐IL‐6 as determined by analysis of NMR relaxation data with the extended model‐free formalism of Lipari and Szabo. To alleviate significant resonance overlap in the HSQC‐type spectra, cell‐free protein synthesis was used to selectively 15N‐label residues, thereby ensuring a complete set of residue‐specific dynamics. The calculated order parameters [square of the generalized model‐free order parameter (S2)] showed significant conformational heterogeneity among clusters of residues in IL‐6. In particular, the N‐terminal region of the long AB‐loop, which corresponds spatially to one of the gp130 receptor binding epitopes (i.e. site III), experiences substantial fluctuations along the conformation of the main chain (S2 = 0.3–0.8) that are not observed at the other two epitopes or in other cytokines. Thus, we postulate that dynamic properties of the AB‐loop are responsible for inhibiting the interaction of IL‐6 with gp130 in the absence of the IL‐6R, and that binding of IL‐6R at site I shifts the dynamic equilibrium to favor interaction with gp130 at site III. In addition, molecular dynamics simulations corroborated the NMR‐derived dynamics, and showed that the BC‐loop adopts different substates that possibly play a role in facilitating receptor assembly.

Metallic-semiconducting junctions create sensing hot-spots in carbon nanotube FET aptasensors near percolation

Easily fabricated random network carbon nanotube field-effect transistors (CNT-FETs) have benefitted from improved separation techniques to deliver CNTs with current formulations providing at least 99% semiconducting tube content. Amongst the most promising applications of this device platform are electronic biosensors, where the network conduction is affected through tethered probes such as aptamers which act as molecular scale electrostatic gates. However, the prevailing assumption that these biosensor devices would be optimized if metallic tubes were entirely eliminated has not been examined. Here, we show that metallic-semiconducting junctions in aptasensors are sensing hotspots and that their impact on sensing is heightened by the CNT networks proximity to percolation. First, we use a biased conducting AFM tip to gate a CNT-FET at the nanoscale and demonstrate that the strongest device response occurs when gating at metallic-semiconducting junctions. Second, we resolve the target sensitivity of an aptasensor as a function of tube density and show heightened sensitivity at densities close to the percolation threshold. We find the strongest sensing response where the 1% of metallic tubes generate a high density of metallic-semiconducting junctions but cannot form a percolated metallic path across the network. These findings highlight the critical role of metallic tubes in CNT-FET biosensor devices and demonstrate that network composition is an important variable to boost the performance of electronic biosensors.

Data on liquid gated CNT network FETs on flexible substrates

This article presents the raw and analyzed data from a set of experiments performed to study the role of junctions on the electrostatic gating of carbon nanotube (CNT) network field effect transistor (FET) aptasensors. It consists of the raw data used for the calculation of junction and bundle densities and describes the calculation of metallic content of the bundles. In addition, the data set consists of the electrical measurement data in a liquid gated environment for 119 different devices with four different CNT densities and summarizes their electrical properties. The data presented in this article are related to research article titled “Metallic-semiconducting junctions create sensing hot-spots in carbon nanotube FET aptasensors near percolation” (doi:10.1016/j.bios.2018.09.021) [1].

Improving odorant chemical class prediction with multi-layer perceptrons using temporal odorant spike responses from drosophila melanogaster olfactory receptor neurons

In this work, we examine the possibility of improving the prediction performance of an olfactory biosensor through the use of temporal spiking data. We present an Artificial Neural Network (ANN), in the form of an optimal hybrid Multi-Layer Perceptron (MLP) system for the classification of chemical odorants from olfactory receptor neuron spike responses of the Drosophila melanogaster fruit fly (DmOrs). The data used in this study contains the responses to 34 odorants from 6 individual DmOrs, of which we exploit the temporal spiking responses of a 500ms odorant stimulus window. We report, for the first time, the difference between the classification performance of the temporal spiking data to an equivalent spontaneous scalar dataset that we have reported previously. We demonstrate that a higher prediction (%) was obtained when using the temporal data, in which a greater number of validation odorants are identified to their correct chemical class. This work presents a novel technique to improve the classification performance of an olfactory biosensor, whilst maintaining a limited sensory array of 6 DmOr receptors.