The dynamics of the β-propeller domain in Kelch protein KLHL40 changes upon nemaline myopathy-associated mutation

Evolutionarily widespread, functionally and structurally diverse and still largely unexplored, Kelch proteins, characterized by the presence of a conserved C-terminal β-propeller, are implicated in a number of diverse fundamental biological functions, including cytoskeletal arrangement, regulation of cell morphology and organization, and protein degradation. Mutations in the genes encoding for Kelch superfamily members are being discovered as the cause of several neuromuscular diseases and cancer. The E528K mutation in Kelch protein KLHL40, which regulates skeletal muscle myogenesis, has been identified as a frequent cause of severe autosomal-recessive nemaline myopathy (NM). We use all-atom molecular dynamics simulations to characterize the dynamic behaviour of the β-propeller of the wild-type protein and identify correlated motions underlying the in vivo functionality. We also modelled the NM-associated mutation and we found that it does not lead to dramatic disruption of the β-propeller architecture; yet, residue 528 is a hub in the correlated motions of the domain, and mutation-induced local structural alterations are propagated to the whole protein, affecting its dynamics and physicochemical properties, which are fundamental for in vivo interaction with partners. Our results indicate that rational design of drugs can be envisioned as a strategy for restoring the internal network of communication and resetting KLHL40 to its physiological state

Assessing the functional and structural stability of the Met80Ala mutant of cytochrome c in dimethylsulfoxide

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and bi-osensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cyto-chrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscop-ically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the mod-ification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physi-ological milieu of cytochrome c

Thermodynamics and Kinetics of Electron Transfer of 2 Electrode-Immobilized Small Laccase from Streptomyces coelicolor

The thermodynamic and kinetic properties for the heterogeneous electron transfer (ET) were measured for the electrode-immobilized small laccase (SLAC) from Streptomyces coelicolor subjected to different electrostatic and covalent protein-electrode linkages, using cyclic voltammetry. Once immobilized electrostatically onto a gold electrode using mixed carboxyl- and hydroxy-terminated alkane-thiolate SAMs or covalently exploiting the same SAM subjected to N-hydroxysuccin-imide+1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS-EDC) chemistry, the SLAC-electrode electron flow occurs through the T1 center. The E°’ values (from +0.2 to +0.1 V vs. SHE at pH 7.0) are lower by more than 0.2 V compared to the protein either in solution or immobilized with different anchoring strategies using uncharged SAMs. For the present electrostatic and covalent binding, this effect can respectively be ascribed to the negative charge of the SAM surfaces and to deletion of the positive charge of Lys/Arg residues due to amide bond formation which both selectively stabilize the more positively charged oxidized SLAC. Observation of enthalpy/entropy compensation within the series indicates that the immobilized proteins experience different reduction-induced solvent reorganization effects. The E°’ values for the covalently attached SLAC are sensitive to three acid base equilibria, with apparent pKa values of pKa1ox =5.1, pKa1red=7.5, pKa2ox=8.4, pKa2red=10.9, pKa2ox=8.9, pKa2red=11.3 possibly involving one residue close to the T1 center and two residues (Lys and/or Arg) along with moderate protein unfolding, respectively. Therefore, the E°’ value of immobilized SLAC turns out to be particularly sensitive to the anchoring mode and me-30 dium conditions

Emerging role of Lon protease as a master regulator of mitochondrial functions

Lon protease is a nuclear-encoded, mitochondrial ATP-dependent protease highly conserved throughout the evolution, crucial for the maintenance of mitochondrial homeostasis. Lon acts as a chaperone of misfolded proteins, and is necessary for maintaining mitochondrial DNA. The impairment of these functions has a deep impact on mitochondrial functionality and morphology. An altered expression of Lon leads to a profound reprogramming of cell metabolism, with a switch from respiration to glycolysis, which is often observed in cancer cells. Mutations of Lon, which likely impair its chaperone properties, are at the basis of a genetic inherited disease named of the cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi

Anti-drug antibody detection with label-free electrolyte-gated organic field-effect transistors

The efficacy of immunotherapy can be undermined by the development of an immune response against a drug/antibody mediated by anti-drug antibodies (ADAs) in treated patients. We present the first label-free EGOFET immunosensor that integrates a biological drug, Nivolumab (Opdivo©), as a specific recognition moiety to quantitatively and selectively detect ADAs against the drug. The limit of detection is 100 fM. This demonstration is a prelude to the detection of ADAs in a clinical setting in the treatment of different pathologies, and it also enables rapid screening of biological drugs for immunogenicity

How to Turn an Electron Transfer Protein into a Redox Enzyme for Biosensing

Cytochrome c is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome c can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response

Experiments and Models for physics learning in primary school

The project “Little scientists in the lab: Experiments & Models for science learning in primary school”, funded by the Ministry of Education and being currently under development in Italy, is addressed to primary school teachers of the Faculty of Science Education and consequently to pupils. This project proposes a “Model-centered Learning Environment” to build pilot activities for teaching and learning science and physics, based on experimental and modeling activities. The approach for teacher training is to assign group tasks that promote learning.We set up a website to support teachers’ school activities to facilitate and promote communication and exchange of materials between teachers and researchers of the University of Modena and Reggio Emilia, as well as between the teachers themselves. In this paper, we illustrate the general features of the project and focus on preliminary results of an in-service training for teachers in fluids and electricity

Modulating the Faradic Operation of All-Printed Organic Electrochemical Transistors by Facile in Situ Modification of the Gate Electrode

Organic electrochemical transistors (OECTs) operated in the faradic regime were shown as outperforming transducers of bioelectric signals in vitro and in vivo. Fabrication by additive manufacturing techniques fosters OECTs as ideal candidates for point-of-care applications, as well as imposes limitations on the choice of materials and their processing conditions. Here, we address the question of how the response of fully printed OECTs depends on gate electrode material. Toward this end, we investigate the redox processes underlying the operation of OECTs under faradic regime, to show OECTs with carbon gate (C-gate) that exhibit no current modulation gate voltages <1.2 V. This is a hallmark that no interference with the faradic operation of the device enabled by redox processes occurs when operating C-gate OECTs in the low-voltage range as label-free biosensors for the detection of electroactive (bio)molecules. To tune the faradic response of the device, we electrodeposited Au on the carbon gate (Au-C-gate), obtaining a device that operates at lower gate voltage values than C-gate OECT. The presence of gold on the gate allowed further modification of the electrical performances by functionalization of the Au-C-gate with different self-assembled monolayers by fast potential-pulse-assisted method. Moreover, we show that the presence in the electrolyte solution of an external redox probe can be used to drive the faradic response of both C- and Au-C-gate OECTs, impacting on the gate potential window that yields effective drain current modulation. The results presented here suggest possible new strategies for controlling the faradic operation regime of OECTs sensors by chemical modification of the gate surface

Thermodynamics and kinetics of reduction and species conversion at a hydrophobic surface for mitochondrial cytochromes c and their cardiolipin adducts

Cytochrome c(cytc) and its adduct with cardiolipin (CL) were immobilized on a hydrophobic SAM-coated electrode surface yielding a construct which mimics the environment experienced by the complex at the inner mitochondrial membrane where it plays a role in cell apoptosis. Under these conditions, both species undergo an equilibrium between a six-coordinated His/His-ligated and a five-coordinated His/- ligated forms stable in the oxidized and in the reduced state, respectively. The thermodynamics of the oxidation-state dependent species conversion were determined by temperature-dependent diffusionless voltammetry experiments. CL binding stabilizes the immobilized reduced His/- ligated form of cytc which was found previously to catalytically reduce dioxygen. Here, this adduct is also found to show pseudoperoxidase activity, catalysing reduction of hydrogen peroxide. These effects would impart CL with an additional role in the cytc-mediated peroxidation leading to programmed cell death. Moreover, Immobilized cytc exchanges electrons more slowly upon CL binding possibly due to changes in solvent reorganization effects at the protein-SAM interface

How Biorecognition Affects the Electronic Properties of Reduced Graphene Oxide in Electrolyte‐Gated Transistor Immunosensors

Ambipolar electrolyte-gated transistors (EGTs) based on reduced graphene oxide (rGO) have been demonstrated as ultra-sensitive and highly specific immunosensors. However, the physics and chemistry ruling the device operation are still not fully unraveled. In this work, the aim is to elucidate the nature of the observed sensitivity of the device. Toward this aim, a physical–chemical model that, coupled with the experimental characterization of the rGO-EGT, allows one to quantitatively correlate the biorecognition events at the gate electrode and the electronic properties of rGO-EGT is proposed. The equilibrium of biorecognition occurring at the gate electrode is shown to determine the apparent charge neutrality point (CNP) of the rGO channel. The multiparametric analysis of the experimental transfer characteristics of rGO-EGT reveals that the recognition events modulate the CNP voltage, the excess carrier density Δn, and the quantum capacitance of rGO. This analysis also explains why hole and electron carrier mobilities, interfacial capacitance, the curvature of the transfer curve, and the transconductances are insensitive to the target concentration. The understanding of the mechanisms underlying the transistor transduction of the biorecognition events is key for the interpretation of the response of the rGO-EGT immunosensors and to guide the design of novel and more sensitive devices