Single molecule conduction of engineered cytochrome b562 bonded to metallic electrodes

Measuring single molecule conductance is a fundamental step in order to realise the basic elements of future electronic circuits. This work describes the use of an engineered electron transfer protein, cytochrome 6562 (cyt 6562), as a single molecule junction point between a gold surface and a metallic tip through defined thiol-metal interactions. Two separate cysteine residues were introduced in the cyt b562 amino acid sequence at strategic positions the single-molecule conductivity of the two double cysteine mutants SH-SA and SH-LA was investigated using atomic force microscopy (AFM), scanning tunnelling microscopy (STM), current-volt age (TV) and current-distance (I-z) experiments. The haem binding properties of the cysteine variants were similar to that of the wild-type cyt 6562 confirming that the mutations had not altered the protein's core properties. AFM and STM studies revealed that the SH-SA and SH-LA molecules bound to gold electrode in defined orientations, dictated by the thiol-pair utilised. A strong and stable interaction between the proteins bearing the thiol groups and a Au(lll) surface was achieved, and a single-molecule conductance of 1 nS w is measured in air. In contrast, the unengineered wild-type cyt 6562 bound much less robustly to the gold surface and the measured conductance was at least one order of magnitude less. Crucially, using electrochemical STM (EC-STM) approaches a change in conductance of the cytochrome over different overpotentials was observed, demonstrating that the molecule can act as an electrochemical gate. The protein became most conducting when the substrate potential was set close to the redox potential of the protein. The electrochemical, I-V and I-z STM measurements sug gested a two-step model for electron transfer. This study illustrates the possibility of exploiting a haem binding nrotein directly adsorbed onto a conducting surface as a nanoelectronic element and offers nw perspectives for future biomolecular electronic circuits

Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase

Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, “nanodiscs”, and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and −300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s(−1). POR was electro-catalytically active while the P450s generated hydrogen peroxide (H(2)O(2)). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products

Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins

Amphipols (APols) are specially designed amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions in the absence of detergent. A8–35, a polyacrylate-based APol, has been grafted with an oligodeoxynucleotide (ODN). The synthesis, purification and properties of the resulting ‘OligAPol’ have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8–35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR/OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile approach to tagging MPs, without modifying them chemically nor genetically, for specific, reversible and targetable immobilization, e.g. for nanoscale applications

Fast electron transfer through a single molecule natively structured redox protein

The electron transfer properties of proteins are normally measured as molecularly averaged ensembles. Through these and related measurements, proteins are widely regarded as macroscopically insulating materials. Using scanning tunnelling microscopy (STM), we present new measurements of the conductance through single-molecules of the electron transfer protein cytochrome b562 in its native conformation, under pseudo-physiological conditions. This is achieved by thiol (SH) linker pairs at opposite ends of the molecule through protein engineering, resulting in defined covalent contact between a gold surface and a platinum–iridium STM tip. Two different orientations of the linkers were examined: a long-axis configuration (SH-LA) and a short-axis configuration (SH-SA). In each case, the molecular conductance could be ‘gated’ through electrochemical control of the heme redox state. Reproducible and remarkably high conductance was observed in this relatively complex electron transfer system, with single-molecule conductance values peaking around 18 nS and 12 nS for the SH-SA and SH-LA cytochrome b562 molecules near zero electrochemical overpotential. This strongly points to the important role of the heme co-factor bound to the natively structured protein. We suggest that the two-step model of protein electron transfer in the STM geometry requires a multi-electron transfer to explain such a high conductance. The model also yields a low value for the reorganisation energy, implying that solvent reorganisation is largely absent

A novel glucuronoxylan hydrolase produced by fermentation is safe as feed additive: toxicology and tolerance in broiler chickens

The current study presents a safety evaluation of a novel glucuronoxylan hydrolase (EC 3.2.1.136) from Bacillus subtilis produced in Bacillus licheniformis. The glucuronoxylan hydrolase preparation did not exhibit irritative potential to the eye and skin when applied in in vitro models. The glucuronoxylan hydrolase preparation was non-mutagenic and non-clastogenic in in vitro tests. Oral administration of the glucuronoxylan hydrolase preparation to rats did not cause any adverse effect in a 90-days subchronic toxicity study. A tolerance study was performed with broiler chickens and confirmed that this glucuronoxylan hydrolase is safe for broiler chickens when fed at the maximum recommended dose, as well as at the 10 times higher dose. In conclusion, there are no safety concerns with using this novel glucuronoxylan hydrolase as a feed additive as it is toxicologically inert and the glucuronoxylan hydrolase is well tolerated by broiler chickens. The beneficial safety evaluation of glucuronoxylan hydrolase is consistent with the fact that this type of enzyme is ubiquitous in nature.info:eu-repo/semantics/publishedVersio

COVID-19 Severity in Multiple Sclerosis: Putting Data Into Context

Background and objectives: It is unclear how multiple sclerosis (MS) affects the severity of COVID-19. The aim of this study is to compare COVID-19-related outcomes collected in an Italian cohort of patients with MS with the outcomes expected in the age- and sex-matched Italian population. Methods: Hospitalization, intensive care unit (ICU) admission, and death after COVID-19 diagnosis of 1,362 patients with MS were compared with the age- and sex-matched Italian population in a retrospective observational case-cohort study with population-based control. The observed vs the expected events were compared in the whole MS cohort and in different subgroups (higher risk: Expanded Disability Status Scale [EDSS] score > 3 or at least 1 comorbidity, lower risk: EDSS score ≤ 3 and no comorbidities) by the χ2 test, and the risk excess was quantified by risk ratios (RRs). Results: The risk of severe events was about twice the risk in the age- and sex-matched Italian population: RR = 2.12 for hospitalization (p < 0.001), RR = 2.19 for ICU admission (p < 0.001), and RR = 2.43 for death (p < 0.001). The excess of risk was confined to the higher-risk group (n = 553). In lower-risk patients (n = 809), the rate of events was close to that of the Italian age- and sex-matched population (RR = 1.12 for hospitalization, RR = 1.52 for ICU admission, and RR = 1.19 for death). In the lower-risk group, an increased hospitalization risk was detected in patients on anti-CD20 (RR = 3.03, p = 0.005), whereas a decrease was detected in patients on interferon (0 observed vs 4 expected events, p = 0.04). Discussion: Overall, the MS cohort had a risk of severe events that is twice the risk than the age- and sex-matched Italian population. This excess of risk is mainly explained by the EDSS score and comorbidities, whereas a residual increase of hospitalization risk was observed in patients on anti-CD20 therapies and a decrease in people on interferon

DMTs and Covid-19 severity in MS: a pooled analysis from Italy and France

We evaluated the effect of DMTs on Covid-19 severity in patients with MS, with a pooled-analysis of two large cohorts from Italy and France. The association of baseline characteristics and DMTs with Covid-19 severity was assessed by multivariate ordinal-logistic models and pooled by a fixed-effect meta-analysis. 1066 patients with MS from Italy and 721 from France were included. In the multivariate model, anti-CD20 therapies were significantly associated (OR = 2.05, 95%CI = 1.39–3.02, p < 0.001) with Covid-19 severity, whereas interferon indicated a decreased risk (OR = 0.42, 95%CI = 0.18–0.99, p = 0.047). This pooled-analysis confirms an increased risk of severe Covid-19 in patients on anti-CD20 therapies and supports the protective role of interferon