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Altres ajuts: CERCA Programme/Generalitat de Catalunya, Fundaluce and "la Caixa" foundations (ID 100010434, agreement LCF/PR/HR19/52160010), Co-financed by the European Union Regional Development Fund within the framework of the ERDF Operational Program of Catalonia 2014-2020 Adrenoceptors are ubiquitous and mediate important autonomic functions as well as modulating arousal, cognition, and pain on a central level. Understanding these physiological processes and their underlying neural circuits requires manipulating adrenergic neurotransmission with high spatio-temporal precision. Here we present a first generation of photochromic ligands (adrenoswitches) obtained via azologization of a class of cyclic amidines related to the known ligand clonidine. Their pharmacology, photochromism, bioavailability, and lack of toxicity allow for broad biological applications, as demonstrated by controlling locomotion in zebrafish and pupillary responses in mice.

Olfactory receptors (ORs) comprise the largest multigene family in the vertebrates. They belong to the class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs), which are the most abundant membrane proteins, having widespread, significant roles in signal transduction in cells, and therefore, they are a major pharmacological target. Moreover, ORs displayed high selectivity and sensitivity towards odorant detection, a characteristic that raised the interest for developing biohybrid sensors based on ORs for the detection of volatile compounds. The transduction of odorant binding into cellular signaling by ORs is not well understood and knowing its mechanism would enable developing new pharmacology and high performance biohybrid electronic sensors. Recent findings suggest that ligand recognition by ORs is determined by the nanoscale alterations of charge distribution in the receptor structure (Ref). However, the electrical characterization of ORs and their response towards ligand binding in bulk experiments is subjected to microscopic models and assumptions [2]. Here, we have directly determined the nanoscale electrical properties of ORs with unprecedented control over the receptor orientation, and their change upon odorant binding, using electrochemical scanning tunneling microscopy (EC-STM) in near-physiological conditions. Recordings of current versus time, distance, and electrochemical potential allows determining the OR impedance parameters and their dependence with odorant binding. The simultaneous measurement of RC equivalent by means of the open-circuit voltage (VOC) allows increasing the electrical sensitivity at the single receptor level for biosensing applications. Our results allow validating OR structural-electrostatic models and their functional activation processes.

Clathrin-mediated endocytosis (CME) is an essential cellular process, which is evolutionarily conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of pharmacological agents that could complement genetics in selectively and reversibly interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor that targets the AP2/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP and demonstrate that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts, thus providing a unique tool for acute and reversible CME modulation in yeast.

Photosynthetic reactions in plants, algae, and cyanobacteria are driven by photosystem I and photosystem II complexes, which specifically reduce or oxidize partner redox biomolecules. Photosynthetic complexes can also bind synthetic organic molecules, which inhibit their photoactivity and can be used both to study the electron transport chain and as herbicides and algicides. Thus, their development, characterization, and sensing bears fundamental and applied interest. Substantial efforts have been devoted to developing photosensors based on photosystem II to detect compounds that bind to the plastoquinone sites of this complex. In comparison, photosystem I based sensors have received less attention and could be used to identify novel substances displaying phytotoxic effects, including those obtained from natural product extracts. We have developed a robust procedure to functionalize gold electrodes with photo- and redox-active photosystem I complexes based on transparent gold and a thiolate self-assembled monolayer, and we have obtained reproducible electrochemical photoresponses. Chronoamperometric recordings have allowed us to measure photocurrents in the presence of the viologen derivative paraquat at concentrations below 100 nM under lock-in operation and a sensor dynamic range spanning six orders of magnitude up to 100 mM. We have modeled their time course to identify the main electrochemical processes and limiting steps in the electron transport chain. Our results allow us to isolate the contributions from photosystem I and the redox mediator, and evaluate photocurrent features (spectral and power dependence, fast transient kinetics) that could be used as a sensing signal to detect other inhibitors and modulators of photosystem I activity.

Visual Abstract, Photopharmacology is a unique approach that through a combination of photochemistry methods and advanced life science techniques allows the study and control of specific biological processes, ranging from intracellular pathways to brain circuits. Recently, a first photochromic channel blocker of anion-selective GABAA receptors, the azobenzene-nitrazepam-based photochromic compound (Azo-NZ1), has been described. In the present study, using patch-clamp technique in heterologous system and in mice brain slices, site-directed mutagenesis and molecular modeling we provide evidence of the interaction of Azo-NZ1 with glycine receptors (GlyRs) and determine the molecular basis of this interaction. Glycinergic synaptic neurotransmission determines an important inhibitory drive in the vertebrate nervous system and plays a crucial role in the control of neuronal circuits in the spinal cord and brain stem. GlyRs are involved in locomotion, pain sensation, breathing, and auditory function, as well as in the development of such disorders as hyperekplexia, epilepsy, and autism. Here, we demonstrate that Azo-NZ1 blocks in a UV-dependent manner the activity of α2 GlyRs (GlyR2), while being barely active on α1 GlyRs (GlyR1). The site of Azo-NZ1 action is in the chloride-selective pore of GlyR at the 2’ position of transmembrane helix 2 and amino acids forming this site determine the difference in Azo-NZ1 blocking activity between GlyR2 and GlyR1. This subunit-specific modulation is also shown on motoneurons of brainstem slices from neonatal mice that switch during development from expressing “fetal” GlyR2 to “adult” GlyR1 receptors.

Manipulation of neuronal activity using two-photon excitation of azobenzene photoswitches with near-infrared light has been recently demonstrated, but their practical use in neuronal tissue to photostimulate individual neurons with three-dimensional precision has been hampered by firstly, the low efficacy and reliability of NIR-induced azobenzene photoisomerization compared to one-photon excitation, and secondly, the short cis state lifetime of the two-photon responsive azo switches. Here we report the rational design based on theoretical calculations and the synthesis of azobenzene photoswitches endowed with both high two-photon absorption cross section and slow thermal back-isomerization. These compounds provide optimized and sustained two-photon neuronal stimulation both in light-scattering brain tissue and in Caenorhabditis elegans nematodes, displaying photoresponse intensities that are comparable to those achieved under one-photon excitation. This finding opens the way to use both genetically targeted and pharmacologically selective azobenzene photoswitches to dissect intact neuronal circuits in three dimensions., Two-photon absorption capacity of azobenzene photoswitches usually comes with a reduction in thermal stability. Here, the authors developed photoswitches with high two-photon sensitivity and enhanced cis isomer thermal lifetime for the control of glutamate receptors.

Summary Kainate receptors (KARs) mediate postsynaptic currents with a key impact on neuronal excitability. However, the molecular determinants controlling KAR postsynaptic localization and stabilization are poorly understood. Here, we exploit optogenetic and single-particle tracking approaches to study the role of KAR conformational states induced by glutamate binding on KAR lateral mobility at synapses. We report that following glutamate binding, KARs are readily and reversibly trapped at glutamatergic synapses through increased interaction with the β-catenin/N-cadherin complex. We demonstrate that such activation-dependent synaptic immobilization of KARs is crucial for the modulation of short-term plasticity of glutamatergic synapses. Thus, the present study unveils the crosstalk between conformational states and lateral mobility of KARs, a mechanism regulating glutamatergic signaling, particularly in conditions of sustained synaptic activity., Graphical Abstract, Highlights • Anchoring of KARs at glutamatergic synapses depends on receptor-glutamate binding • KARs activation/desensitization promotes receptors trapping at glutamatergic synapses • N-cadherins mediate the KAR activation/desensitization-dependent anchoring at synapses • Synaptic trapping of desensitized KARs affects short-term synaptic plasticity, Polenghi et al. report that following their activation, kainate receptors (KARs) are readily trapped at glutamatergic synapses in the open/desensitized state. This mechanism defines the number of ready-to-be-activated KARs at synapses, with direct impacts on short-term synaptic plasticity.

Altres ajuts: CERCA Programme/Generalitat de Catalunya Aquest article té una correcció a 10.1039/d0sc90189j The spatiotemporal control of cellular dynamic processes has great fundamental interest but lacks versatile molecular tools. Dynamin is a key protein in endocytosis and an appealing target to manipulate cell trafficking using patterns of light. We have developed the first photoswitchable small-molecule inhibitors of endocytosis (dynazos), by a stepwise design of the photochromic and pharmacological properties of dynasore, a dynamin inhibitor. We have characterized their photochromism with UV-visible and transient absorption spectroscopy and their biological activity using fluorescence microscopies and flow cytometry. Dynazos are water-soluble, cell permeable, and photostable, and enable fast, single-wavelength photoswitchable inhibition of clathrin-mediated endocytosis at micromolar concentration.

Correction for ‘Photoswitchable dynasore analogs to control endocytosis with light’ by Núria Camarero et al., Chem. Sci., 2020, 11, 8981–8988, DOI: 10.1039/d0sc03820b.

Glycine receptors (GlyRs) are indispensable to maintain excitatory/inhibitory balance in neuronal circuits controlling reflex and rhythmic motor behaviors. Here we have developed Glyght, the first GlyR ligand controlled with light. It is selective over other cys-loop receptors, active in vivo, and displays an allosteric mechanism of action. The photomanipulation of glycinergic neurotransmission opens new avenues to understand inhibitory circuits in intact animals, and to develop drug-based phototherapies.

The physiological activity of proteins is often studied with loss-of-function genetic approaches, but the corresponding phenotypes develop slowly and can be confounding. Photopharmacology allows direct, fast, and reversible control of endogenous protein activity, with spatiotemporal resolution set by the illumination method. Here, we combine a photoswitchable allosteric modulator (alloswitch) and 2-photon excitation using pulsed near-infrared lasers to reversibly silence metabotropic glutamate 5 (mGlu5) receptor activity in intact brain tissue. Endogenous receptors can be photoactivated in neurons and astrocytes with pharmacological selectivity and with an axial resolution between 5 and 10 μm. Thus, 2-photon pharmacology using alloswitch allows investigating mGlu5-dependent processes in wild-type animals, including synaptic formation and plasticity, and signaling pathways from intracellular organelles. © 2019 National Academy of Sciences. All rights reserved., ACKNOWLEDGMENTS. We thank Jordi Hernando (Autonomous University of Barcelona) for useful discussions on 2-photon excitation; Pere Català (Utrecht University) for help with GCaMP; Francisco Ciruela (University of Barcelona) for mGlu5-eYFP plasmid; Erin Schuman and Stephan Junek (Max Planck Institute for Brain Research, Frankfurt) for preliminary 2-photon excitation experiments; and Ashraf Muhaisen (University of Barcelona) for help with slicing. This research received funding from European Union Research and Innovation Programme Horizon 2020 [Human Brain Project SGA2 Grant Agreement 785907 (WaveScalES)], European Research ERA-Net SynBio programme (Modulightor project), and financial support from Agency for Management of University and Research Grants/Generalitat de Catalunya (CERCA Programme; 2017-SGR-1442 project), Fonds Européen de Développement Économique et Régional (FEDER) funds, Ministry of Economy and Competitiveness (MINECO)/FEDER (Grant CTQ2016-80066-R), and the Fundaluce foundation. S.P. was supported by an FI fellowship from the Agency for Management of University and Research Grants/Generalitat de Catalunya (2014FI_B2 00160). H.L. was supported by an Institute for Bioengineering of Catalonia Severo Ochoa International PhD Programme fellowship from MINECO. M.B. was supported by a H2020-MSCA-IF Reintegration Grant. K.E.P. receives support from NIH/National Institute of Neurological Disorders and Stroke Grant R01NS099254 and NSF Biophotonics Grant 1604544. E.S. receives support from MINECO (Grant SAF2016-7426).

Remote control of physiological functions with light offers the promise of unveiling their complex spatiotemporal dynamics in vivo, and enabling highly focalized therapeutic interventions with reduced systemic toxicity. Optogenetic methods have been implemented in the heart, but the need of genetic manipulation jeopardizes clinical applicability. This study aims at developing, testing and validating the first light-regulated drug with cardiac effects, in order to avoid the requirement of genetic manipulation offered by optogenetic methods. A M2 muscarinic acetylcholine receptors (mAChRs) light-regulated drug (PAI) was designed, synthesized and pharmacologically characterized. The design was based on the orthosteric mAChRs agonist Iperoxo, an allosteric M2 ligand, and a photoswitchable azobenzene linker. PAI can be reversibly photoisomerized between cis and trans configurations under ultraviolet (UV) and visible light, respectively, and it reversibly photoswitches the activity of M2 muscarinic acetylcholine receptors. We have evaluated in vitro photoresponses using a calcium imaging assay in genetically unmodified receptors overexpressed in mammalian cells. Furthermore, using this new chemical tool, we demonstrate for the first time photoregulation of cardiac function in vivo in wildtype frog tadpoles and in rats with a method that does not require genetic manipulation. Such a new approach may enable enhanced spatial and temporal selectivity for cardiovascular drugs.

The efficacy and tolerability of systemically administered anticancer agents are limited by their off-target effects. Precise spatiotemporal control over their cytotoxic activity would allow improving chemotherapy treatments, and light-regulated drugs are well suited to this purpose. We have developed phototrexate, the first photoswitchable inhibitor of the human dihydrofolate reductase (DHFR), as a photochromic analogue of methotrexate, a widely prescribed chemotherapeutic drug to treat cancer and psoriasis. Quantification of the light-regulated DHFR enzymatic activity, cell proliferation, and in vivo effects in zebrafish show that phototrexate behaves as a potent antifolate in its photoactivated cis configuration and that it is nearly inactive in its dark-relaxed trans form. Thus, phototrexate constitutes a proof-of-concept to design light-regulated cytotoxic small molecules and a step forward to develop targeted anticancer photochemotherapies with localized efficacy and reduced adverse effects.

Despite the importance of electron transfer (ET) between redox proteins in photosynthesis and respiration, the inter-protein ET rate between redox partner proteins has never been measured as a function of their separation in aqueous solution. Here we use electrochemical tunneling spectroscopy to show that the current between two protein partners decays along more than 10 nm in the solution. Molecular dynamics simulations reveal a reduced ionic density and extended electric field in the volume confined between the proteins. The distance-decay factor and the calculated local barrier for ET are regulated by the electrochemical potential applied to the proteins. Redox partners could use electrochemically gated, long distance ET through the solution in order to conciliate high ET specificity with weak binding, thus keeping high turnover rates in the crowded environment of cells., The project was funded by CERCA, FEDER, and Networking Biomedical Research Center (CIBER), Spain. CIBER is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and the Instituto de Salud Carlos III, with the support of the European Regional Development Fund. This work was financially supported by the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (2017 SGR 1442, 2014 SGR 1442, and 2017 SGR 1189), by the Andalusian Government (BIO198), and the projects CTQ2016–80066-R, CTQ2015-66194-R, CTQ2017-85496-P and Spanish Structures of Excellence Maria de Maeztu, MDM2017-0767, and BFU2015-71017-P/BMC awarded by the Spanish Ministry of Economy and Competitiveness. A.N.-H. thanks the Generalitat de Catalunya-AGAUR for a FI scholarship. MINECO/ICTI2013-2016/CTQ2015-66194-R MINECO/ICTI2013-2016/BFU2015-71017-P MICIU/ICTI2017-2020/CTQ2017-85496-P MICIU/ICTI2017-2020/MDM2017-0767

Bioelectronics moves toward designing nanoscale electronic platforms that allow in vivo determinations. Such devices require interfacing complex biomolecular moieties as the sensing units to an electronic platform for signal transduction. Inevitably, a systematic design goes through a bottom-up understanding of the structurally related electrical signatures of the biomolecular circuit, which will ultimately lead us to tailor its electrical properties. Toward this aim, we show here the first example of bioengineered charge transport in a single-protein electrical contact. The results reveal that a single point-site mutation at the docking hydrophobic patch of a Cu-azurin causes minor structural distortion of the protein blue Cu site and a dramatic change in the charge transport regime of the single-protein contact, which goes from the classical Cu-mediated two-step transport in this system to a direct coherent tunneling. Our extensive spectroscopic studies and molecular-dynamics simulations show that the proteins' folding structures are preserved in the single-protein junction. The DFT-computed frontier orbital of the relevant protein segments suggests that the Cu center participation in each protein variant accounts for the different observed charge transport behavior. This work is a direct evidence of charge transport control in a protein backbone through external mutagenesis and a unique nanoscale platform to study structurally related biological electron transfer.

Understanding how charges move through and between biomolecules is a fundamental question that constitutes the basis for many biological processes. On the other hand, it has potential applications in the design of sensors based on biomolecules and single molecule devices. In this review we introduce the study of the electron transfer (ET) process in biomolecules, providing an overview of the fundamental theory behind it and the different experimental approaches. The ET in proteins is introduced by reviewing a complete electronic characterization of a redox protein (azurin) using electrochemical scanning tunnelling microscopy (ECSTM). The ET process in DNA is overviewed and results from different experimental approaches are discussed. Finally, future directions in the study of the ET process in biomolecules are introduced as well as examples of possible technological applications.

and single-molecule char-acteristics. Finding out whether such switching events can also be observed in redox proteins, and what their contribu-tion to the average electrochemical behavior is, could help to understand the mechanisms behind biological ET processes, as well as lead to the designing of reliable devices based on individual or small assemblies of molecules. In single-azurin studies, deviations from the average redox behavior have been observed in current–time traces of ‘wired’ junctions by ECSTM.

A lipase was immobilized on transparent agarose microspheres and genetically engineered to specifically anchor photochromic molecules into its catalytic site. Several combinations of azobenzene and spiropyran groups were conjugated to cysteines introduced at different positions near the active center. Light modulated the catalytic properties of the resulting solid bioconjugates, and such modulation depended on both the nature of the photochromic compound and the anchoring position. Covalent anchoring of azobenzene derivatives to the residue 295 of the lipase 2 from Bacillus thermocathenolatus triggered lipase preference for the S isomer under UV light, whereas visible light promoted preference for the R isomer. Molecular dynamics simulations indicate that conjugating photochromic compounds into the catalytic cavity allows manipulating the steric hindrance and binding energy of the substrates, leading to an enantioselective molecular fit in certain cases. Using this approach, we report for the first time the control of enzyme properties using light in the solid phase., We are grateful for financial support from the Human Frontier Science Program through a Career Development Award (CDA022/2006), from the European Research Council through the “Opticalbullet” Starting Grant (ERC-StG210355/2007), from the European Commission through the “Focus” ICT-FET Grant (FP7-ICT-2009-270483), from the Ministry of Education through Grants CTQ2008-06160 and CTQ2011-25871, a “Juan de la Cierva” research associate fellowship, and FPI and FPU fellowships, and from the RecerCaixa and Marató de TV3 foundations.

Control of membrane traffic: Photoswitchable inhibitors of protein-protein interactions were applied to photoregulate clathrin-mediated endocytosis (CME) in living cells. Traffic light (TL) peptides acting as "stop" and "go" signals for membrane traffic can be used to dissect the role of CME in receptor internalization and in cell growth, division, and differentiation.

Light-regulated drugs allow remotely photoswitching biological activity and enable plausible therapies based on small molecules. However, only freely diffusible photochromic ligands have been shown to work directly in endogenous receptors and methods for covalent attachment depend on genetic manipulation. Here we introduce a chemical strategy to covalently conjugate and photoswitch the activity of endogenous proteins and demonstrate its application to the kainate receptor channel GluK1. The approach is based on photoswitchable ligands containing a short-lived, highly reactive anchoring group that is targeted at the protein of interest by ligand affinity. These targeted covalent photoswitches (TCPs) constitute a new class of light-regulated drugs and act as prosthetic molecules that photocontrol the activity of GluK1-expressing neurons, and restore photoresponses in degenerated retina. The modularity of TCPs enables the application to different ligands and opens the way to new therapeutic opportunities., We are grateful to G. Swanson (Northwestern University Feinberg School of Medicine) for the GluK1-2b receptor clone and advice for DRG neuronal culture, and to M. Mayer (National Institutes of Health) for GluK1 S1S2 plasmid and purification protocol. We also thank E. Vázquez (Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona) for providing the Origami-B (DE3) strain, and O. Seria, J.A. del Rio, G. Callejo and X. Gasull for help with DRG neuron cultures. We are grateful to D. Soto and A. Llobet for helpful discussions. MS was performed at the IRB Barcelona Mass Spectrometry Core Facility, which actively participates in the BMBS European COST Action BM 1403 and is a member of Proteored, PRB2-ISCIII, supported by grant PRB2 (IPT13/0001-ISCIII-SGEFI / FEDER). We want to thank M. Vilaseca and M. Vilanova for technical support with MS. We acknowledge financial support from the RecerCaixa foundation (2010ACUP00378); the Marató de TV3 Foundation (grants 110231 and 111531); the Human Brain Project (HBP SGA 1), the Catalan government (2012FI_B 01122, 2014SGR-1251, 2014SGR-00109 and 2009SGR-1072); the Spanish Government (SAF2012-36375, CTQ2013-43892R and CTQ2014-57020-R); the Ramón Areces foundation and the ERANET Neuron LIGHTPAIN and SynBio MODULIGHTOR projects.

Understanding how molecular conductance depends on voltage is essential for characterizing molecular electronics devices. We reproducibly measured current-voltage characteristics of individual redox-active proteins by scanning tunneling microscopy under potentiostatic control in both tunneling and wired configurations. From these results, transition voltage spectroscopy (TVS) data for individual redox molecules can be calculated and analyzed statistically, adding a new dimension to conductance measurements. The transition voltage (TV) is discussed in terms of the two-step electron transfer (ET) mechanism. Azurin displays the lowest TV measured to date (0.4 V), consistent with the previously reported distance decay factor. This low TV may be advantageous for fabricating and operating molecular electronic devices for different applications. Our measurements show that TVS is a helpful tool for single-molecule ET measurements and suggest a mechanism for gating of ET between partner redox proteins.

Bistable molecules that behave as switches in solution have long been known. Systems that can be reversibly converted between two stable states that differ in their physical properties are particularly attractive in the development of memory devices when immobilized in substrates. Here, we report a highly robust surface-confined switch based on an electroactive, persistent organic radical immobilized on indium tin oxide substrates that can be electrochemically and reversibly converted to the anion form. This molecular bistable system behaves as an extremely robust redox switch in which an electrical input is transduced into optical as well as magnetic outputs under ambient conditions. The fact that this molecular surface switch, operating at very low voltages, can be patterned and addressed locally, and also has exceptionally high long-term stability and excellent reversibility and reproducibility, makes it a very promising platform for non-volatile memory devices.

Photoswitched tethered ligands (PTLs) can be used to remotely control protein function with light. We have studied the geometric and conformational factors that determine the efficacy of PTL gating in the ionotropic glutamate receptor iGluR6 using a family of photoiosomerizable MAG (maleimide-azobenzene-glutamate) PTLs that covalently attach to the clamshell ligand-binding domain. Experiments and molecular dynamics simulations of the modified proteins show that optical switching depends on 2 factors: ( i ) the relative occupancy of the binding pocket in the 2 photoisomers of MAG and ( ii ) the degree of clamshell closure that is possible given the disposition of the MAG linker. A synthesized short version of MAG turns the channel on in either the cis or trans state, depending on the point of attachment. This yin/yang optical control makes it possible for 1 wavelength of light to elicit action potentials in one set of neurons, while deexciting a second set of neurons in the same preparation, whereas a second wavelength has the opposite effect. The ability to generate opposite responses with a single PTL and 2 versions of a target channel, which can be expressed in different cell types, paves the way for engineering opponency in neurons that mediate opposing functions.

Although the identity and interactions of signaling proteins have been studied in great detail, the complexity of signaling networks cannot be fully understood without elucidating the timing and location of activity of individual proteins. To do this, one needs a means for detecting and controlling specific signaling events. An attractive approach is to use light, both to report on and control signaling proteins in cells, because light can probe cells in real time with minimal damage. Although optical detection of signaling events has been successful for some time, the development of the means for optical control has accelerated only recently. Of particular interest is the development of chemically engineered proteins that are directly sensitive to light.

The analysis of cell signaling requires the rapid and selective manipulation of protein function. We have synthesized photoswitches that covalently modify target proteins and reversibly present and withdraw a ligand from its binding site due to photoisomerization of an azobenzene linker. We describe here the properties of a glutamate photoswitch that controls an ion channel in cells. Affinity labeling and geometric constraints ensure that the photoswitch controls only the targeted channel, and enables spatial patterns of light to favor labeling in one location over another. Photoswitching to the activating state places a tethered glutamate at a high (millimolar) effective local concentration near the binding site. The fraction of active channels can be set in an analog manner by altering the photostationary state with different wavelengths. The bistable photoswitch can be turned on with millisecond-long pulses at one wavelength, remain on in the dark for minutes, and turned off with millisecond long pulses at the other wavelength, yielding sustained activation with minimal irradiation. The system provides rapid, reversible remote control of protein function that is selective without orthogonal chemistry.

A new azobenzene-based photoswitch, 2, has been designed to enable optical control of ionotropic glutamate receptors in neurons via sensitized two-photon excitation with NIR light. In order to develop an efficient and versatile synthetic route for this molecule, a modular strategy is described which relies on the use of a new linear fully protected glutamate derivative stable in basic media. The resulting compound undergoes one-photon trans-cis photoisomerization via two different mechanisms: direct excitation of its azoaromatic unit and irradiation of the pyrene sensitizer, a well-known two-photon sensitive chromophore. Moreover, 2 presents large thermal stability of its cis isomer, in contrast to other two-photon responsive switches relying on the intrinsic nonlinear optical properties of push-pull substituted azobenzenes. As a result, the molecular system developed herein is a very promising candidate for evoking large photoinduced biological responses during the multiphoton operation of neuronal glutamate receptors with NIR light, which require accumulation of the protein-bound cis state of the switch upon repeated illumination.

The structural basis of the low reorganization energy of cupredoxins has long been debated. These proteins reconcile a conformationally heterogeneous and exposed metal-chelating site with the highly rigid copper center required for efficient electron transfer. Here we combine single-molecule mechanical unfolding experiments with statistical analysis and computer simulations to show that the metal-binding region of apo-azurin is mechanically flexible and that high mechanical stability is imparted by copper binding. The unfolding pathway of the metal site depends on the pulling residue and suggests that partial unfolding of the metal binding site could be facilitated by the physical interaction with certain regions of the redox protein. We are grateful to A. Donaire and I. Díez-Pérez for discussions, and to the Catalan government (grant 2014SGR-1251), the Spanish government (grant CTQ2013-43892R) and the European Research Council (PELE ERC-2009-Adg 25027) for financial support.

We have measured in situ the electronic conductance spectra of the passive film formed on an Fe electrode immersed in a borate buffer solution using electrochemical tunneling spectroscopy (ECTS) and electrochemical impedance spectroscopy (EIS) techniques, and we have followed their changes as the electrode is electrochemically oxidized and reduced. We demonstrate that pre-passive Fe(II) oxide and the passive Fe(II)/Fe(III) film, behave as p- and n-type semiconductors, respectively and that their reversible inter-conversion is mediated by the availability of free charge carriers on the electrode surface. ECTS spectra have been also modeled to obtain the main electrochemical kinetic parameters of the electron transfer through both p-Fe(II) and n-Fe(III) oxides at different sample potentials and pHs values. We find that the electronic energy barrier in the oxide and its dependence with electrode potential and solution pH, determine the reactivity and passivity of iron. Keywords: Electrochemical tunneling spectroscopy, Fe passivity, Electronic energy barriers, pH effect on passivity

We describe a methodology to perform reliable tunneling spectroscopy in electrochemical media. Sequential in situ tunneling spectra are recorded while the electrochemical potential of the electrode is scanned. Spectroscopic data are presented as conductance maps or conductograms that show the in situ electronic structure of an electrode surface while it undergoes an electrochemical reaction. The conductance map or conductogram represents the redox fingerprint of an electrode/liquid interface in a specific medium and can serve to predict its electrochemical behavior in a quantitative energy scale. The methodology is validated studying the reversible oxidation and passivity of an iron electrode in borate buffer, and we describe the main quantitative information that can be extracted concerning the semiconducting properties of the Fe passive film. This methodology is useful to study heterogeneous catalysis, electrochemical sensing and bioelectronic systems.

The understanding of metal passivation has greatly improved in the recent years due to the development of in situ experimental techniques. It is now possible to characterize at the nanoscale the chemical composition, structure and electronic properties of oxide films that grow on the surface of metals, while keeping them in solution and under potentiostatic control. This knowledge is allowing to elucidate the mechanisms of metal passivation and is providing great advances in the characterization of other dynamic, complex processes like passivity breakdown induced by chloride.

Despite its tremendous scientific and economic impact, the mechanism that triggers metal passive film breakdown in the presence of aggressive ions remains under discussion. We have studied the iron passive film in chloride media using X-ray photoelectron spectroscopy (XPS), electrochemical impedance spectroscopy and electrochemical tunneling spectroscopy (ECTS). Ex situ XPS reveal that the film consists exclusively of an Fe(III) oxide without chloride content. In situ ECTS has been used to build up conductance maps of the Fe electrode during its electrochemical oxidation in a borate buffer solution and its breakdown when the film is grown in the presence of chloride. This conductograms provide direct and in situ experimental evidence of chloride-induced surface states within the band gap of the oxide film (∼3.3 eV). These states enable new charge exchange pathways that allow hole capture at the surface of the n-type Fe(III) oxide. The blocking of VB processes that occurs in the iron passive film is no longer present in chloride media, and electrode corrosion can proceed through these new states. We propose a simple 3-step mechanism for the process, in which chloride anions form an oxidizing Fe(II) surface intermediate but do not participate directly in the reaction. Keywords: Electrochemical tunneling spectroscopy, Electronic band structure, Fe passive film, Aqueous chloride corrosion, Semiconductor decomposition, Interface states

The precise regulation of protein activity is fundamental to life. The allosteric control of an active site by a remote regulatory binding site is a mechanism of regulation found across protein classes, from enzymes to motors to signaling proteins. We describe a general approach for manipulating allosteric control using synthetic optical switches. Our strategy is exemplified by a ligand-gated ion channel of central importance in neuroscience, the ionotropic glutamate receptor (iGluR). Using structure-based design, we have modified its ubiquitous clamshell-type ligand-binding domain to develop a light-activated channel, which we call LiGluR. An agonist is covalently tethered to the protein through an azobenzene moiety, which functions as the optical switch. The agonist is reversibly presented to the binding site upon photoisomerization, initiating clamshell domain closure and concomitant channel gating. Photoswitching occurs on a millisecond timescale, with channel conductances that reflect the photostationary state of the azobenzene at a given wavelength. Our device has potential uses not only in biology but also in bioelectronics and nanotechnology.

The oxidation and reduction processes of both polycrystalline tin and Sn(100) single crystals are investigated in borate buffer solution of pH 7.5 using cyclic voltammetry and in-situ characterizat...

The electrochemical behavior of tin polycrystals in borate buffer solutions at pH 7.5 was systematically investigated using cyclic voltammetry, EC-STM and electrochemical impedance measurements. A systematic shift to more negative values of the potentials corresponding either to the major reduction peak either to the hydrogen evolution was measured when the anodization potential used for film growth was increased. A discussion about the different oxides there formed was included and a reassignation of tin oxidation and tin oxides reduction processes was given. Neste trabalho, foi estudado o comportamento eletroquímico de estanho policristalino em soluções de tampão borato com pH 7.5, utilizando voltametria cíclica, eletroquímica com microscopia de varredura (EC-STM) e espectroscopia de impedância eletroquímica. Os potenciais do pico principal de redução do óxido e de geração de hidrogênio deslocaram-se de forma sistemática para valores mais negativos quando se aumentou o potencial anódico de formação do filme. Foi discutida a natureza dos diferentes óxidos formados e também foi dada nova atribuição aos processos de oxidação do estanho e de redução de seus óxidos.

Since hydrophilic matrices were proposed for controlled drug delivery, many polymeric excipients have been studied to make drug release fit the desired profiles. It has been pointed out that lambda-carrageenan, a sulfated polymer from algae, can suitably control the release rate of basic drugs from hydrophilic matrices. Furthermore, the relevance of hydrophobic interactions in drug-polymer aqueous systems has already been demonstrated, although no references to morphological features as well as to the kinetics of the interaction complexes formation have been published to date. In this work, we propose a method to monitor the topographical evolution of the interaction between lambda-carrageenan and dexchlorpheniramine maleate, in order to determine how the release profiles can be so easily controlled. For this purpose, solutions of both polymer and drug were prepared at very low concentration. Solutions were mixed and small volumes were taken every hour for over a period of 24 h and subsequently analyzed. The characterization technique used, atomic force microscopy, provides a high resolution, allowing plotting of three-dimensional images of the sample morphology within the nanometric scale. The results demonstrate that lambda-carrageenan is able to nanoencapsulate spontaneously dexchlorpheniramine maleate molecules, which offers the possibility of controlling the release rate of the drug with no need of complex technological processes. Moreover, this work demonstrates the suitability of atomic force microscopy for the specific case of the on-time monitoring of interaction processes that occur in pharmaceutical systems.

Since hydrophilic matrices were proposed for controlled drug delivery, many polymeric excipients have been studied in order to make drug release fit the desired profiles. It has been pointed out that lambda-carrageenan, a sulphated polymer from algae, can suitably control the release rate of basic drugs from hydrophilic matrices with no need for complex technological processes. In this work, we propose a method to monitor morphologically the interaction between lambda-carrageenan and dexchlorpheniramine maleate (D-CPM), in order to find out how the release profiles can be so easily controlled. To this end, solutions of both polymer and drug were prepared at very low concentration. Solutions were mixed and samples were taken every hour over a period of 20 h. The characterization technique employed, atomic force microscopy (AFM), provides a high resolution, allowing to show the three-dimensional morphology of the samples within the nanometric scale. The results demonstrate that lambda-carrageenan is able to nanoencapsulate spontaneously D-CPM molecules, which offers the possibility to easily control the release rate of the drug. This work has moreover demonstrated the suitability of AFM for the specific case of the on-time monitoring of interaction processes that happen in pharmaceutical systems.

gamma-Zein, a maize storage protein with an N-terminal proline-rich repetitive domain (gamma-ZNPRD), is located at the periphery of protein bodies. This domain appears to be indispensable for the aggregation of the protein on the surface of the organelle. The peptide (VHLPPP)8, spanning the gamma-ZNPRD, adopts a polyproline II (PPII) conformation that gives an amphipathic helix different from the alpha-helix. We used atomic force microscopy to study the surface organisation of the octamer, and transmission electron microscopy to visualise aggregates of the peptide in aqueous solution. We consider two self-assembly patterns that take account of the observed features. The micellar one fits best with the experimental results presented. Moreover, we found that this peptide has properties associated with surfactants, and form micelles in solution. This spontaneous amphipathic arrangement of the gamma-ZNPRD suggests a mechanism of gamma-zein deposition inside maize protein bodies.

SnO 2 crystalline semiconductor nanoparticles have been studied by scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) in order to analyze the change of conductance spectra in an oxidizing or reducing atmosphere. We measured tunneling current versus bias voltage and obtained STM images in different bias voltage conditions for SnO 2 nanoparticles placed on the surface of different gas sensor layers. There was a reduction of surface bandgap and a displacement of the gap to positive bias potentials in a reducing environment, but we observed an increase of surface bandgap and a displacement to negative bias potentials in an oxidizing atmosphere. The presented methodology of study becomes an innovative powerful tool for the development and understanding of semiconductor gas sensors.

Some observations made on the nature and distribution of monolayer (elementary) steps on the (100) cleavage faces of MgO single crystals by atomic force microscopy are presented and discussed. The following types of patterns of monolayer steps are described: (1) trains of steps, (2) steps terminating on the cleaved surface at the emergence points of screw dislocations, and (3) localized pinning of advancing steps at random sites (probably at the emergence points of edge dislocations). It is shown that: (1) the origins of emergence points of monolayer steps are devoid of hollow cores due to a small Burgers vector of dislocations and (2) the minimum distance between two emerging steps due to screw dislocations and between two pinning centres due to edge dislocations depends on their sign, and is determined by the mutual interaction between neighbouring dislocations.

A method is presented for simultaneously producing porous silicon and depositing platinum on silicon from a platinum and fluoride solution operating at the rest potential. The resulting layers display visible photoluminescence. Quantitative electrochemical measurements demonstrate that the platinum ions act as an oxidizing agent for silicon, and that the silicon oxidation reaction proceeds in the porous silicon regime when the solution parameters are properly chosen.

Highly oriented thin films of tetrathiafulvalene tetracyanoquinodimethane (TTF-TCNQ) and of p-nitrophenyl nitronyl nitroxide (p-NPNN) have been grown on alkali halide substrates by vapor deposition techniques. Their surfaces reveal clearly differentiated morphologies, as measured with tapping mode atomic force microscopy (TMAFM), exhibiting different kinds of defects. However, the films order in similar layered molecular structures parallel to the substrate plane.

Some experimental observations made on the nature and distribution of nearly circular and polygonal closed steps, and arrays of semicircular steps arranged between two multilayer steps on the (100) cleavage planes of MgO single crystals by atomic force microscopy are described. The results show that: (1) the formation of disc-shaped elevations and depressions, and series of semicircular steps takes place by a mechanism similar to the Frank–Read source [Phys. Rev. 79 (1950) 722]; (2) the diameters of disc-shaped structures are related with shear stresses of the order of the yield stress of the crystal; and (3) with an increase in the size of closed steps, their rounded shape changes to polygonal due to a lower edge free energy of the 〈010〉 and 〈011〉 steps compared to that of steps of other orientations.

We have studied by means of tapping mode atomic force microscopy ex situ grown surfaces of highly-oriented α-phase thin films of the molecular organic radical p-nitrophenyl nitroxyl nitroxide. The films are grown by thermal evaporation of the precursor radical in high vacuum on glass and NaCl(001) substrates. The surfaces reveal a complex nanometer-scale morphology formed by a random distribution of dislocations identified as spirals (screw dislocations) of opposite sign interacting in pairs. Each spiral emerges from a hollow core with a diameter of ca 15–20 nm. The dislocation density is rather high due to the way in which the films grow under the given experimental conditions. The out-of-plane growth of the films is governed by the Frank–Read source mechanism.

This is an open access article published under an ACS AuthorChoice License. See Standard ACS AuthorChoice/Editors' Choice Usage Agreement - https://pubs.acs.org/page/policy/authorchoice_termsofuse.html Synthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution.

The development of photochromic and photoswitchable ligands for ion channels and receptors has made important contributions to optopharmacology and optogenetic pharmacology. These compounds provide new tools to study ion channel proteins and to understand their function and pathological implications. Here, we describe the design, operation, and applications of the available photoswitches, with special emphasis on ligand- and voltage-gated channels.

This is an open access article published under an ACS AuthorChoice License. See Standard ACS AuthorChoice/Editors' Choice Usage Agreement - https://pubs.acs.org/page/policy/authorchoice_termsofuse.html The adenosinergic system operates through G protein-coupled adenosine receptors, which have become promising therapeutic targets for a wide range of pathological conditions. However, the ubiquity of adenosine receptors and the eventual lack of selectivity of adenosine-based drugs have frequently diminished their therapeutic potential. Accordingly, here we aimed to develop a new generation of light-switchable adenosine receptor ligands that change their intrinsic activity upon irradiation, thus allowing the spatiotemporal control of receptor functioning (i.e., receptor activation/inactivation dependent on location and timing). Therefore, we synthesized an orthosteric, photoisomerizable, and nonselective adenosine receptor agonist, nucleoside derivative MRS5543 containing an aryl diazo linkage on the N substituent, which in the dark (relaxed isomer) behaved as a full adenosine A receptor (AR) and partial adenosine A receptor (AR) agonist. Conversely, upon photoisomerization with blue light (460 nm), it remained a full AR agonist but became an AR antagonist. Interestingly, molecular modeling suggested that structural differences encountered within the third extracellular loop of each receptor could modulate the intrinsic, receptor subtype-dependent, activity. Overall, the development of adenosine receptor ligands with photoswitchable activity expands the pharmacological toolbox in support of research and possibly opens new pharmacotherapeutic opportunities.

Background Neurons signal to each other and to non-neuronal cells as those in muscle or glands, by means of the secretion of neurotransmitters at chemical synapses. In order to dissect the molecular mechanisms of neurotransmission, new methods for directly and reversibly triggering neurosecretion at the presynaptic terminal are necessary. Here we exploit the calcium permeability of the light-gated channel LiGluR in order to reversibly manipulate cytosolic calcium concentration, thus controlling calcium-regulated exocytosis. Methods Bovine chromaffin cells expressing LiGluR were stimulated with light. Exocytic events were detected by amperometry or by whole-cell patch-clamp to quantify membrane capacitance and calcium influx. Results Amperometry reveals that optical stimulation consistently triggers exocytosis in chromaffin cells. Secretion of catecholamines can be adjusted between zero and several Hz by changing the wavelength of illumination. Differences in secretion efficacy are found between the activation of LiGluR and native voltage-gated calcium channels (VGCCs). Our results show that the distance between sites of calcium influx and vesicles ready to be released is longer when calcium influx is triggered by LiGluR instead of native VGCCs. Conclusion LiGluR activation directly and reversibly increases the intracellular calcium concentration. Light-gated calcium influx allows for the first time to control calcium-regulated exocytosis without the need of applying depolarizing solutions or voltage clamping in chromaffin cells. General significance LiGluR is a useful tool to study the secretory mechanisms and their spatiotemporal patterns in neurotransmission, and opens a window to study other calcium-dependent processes such as muscular contraction or cell migration.