Staff member


Pau Gorostiza Langa

Group Leader / ICREA Research Professor
Nanoprobes and Nanoswitches
pgorostiza@ibecbarcelona.eu
+34 934 020 208
CV Summary

Pau Gorostiza graduated in physics at the Universitat de Barcelona (UB), where he also obtained his Ph.D. (European Doctorate) in the field of semiconductor electrochemistry. He also worked at the microscopy facility of the UB, where he gained experience in AFM and STM of biological samples, as well as in nanotechnology applied to materials science. He has visited the CNRS and the Université Pierre et Marie Curie in Paris (France), and the University of California at Berkeley (USA). His recent works include the development of optical switches for remotely controlling neuronal activity. He obtained a Young Biomedical Investigator Award of the Francisco Cobos Foundation, a Career Development Award of the Human Frontier Science Program (HFSP) and a Starting Grant of the European Research Council (ERC). He is currently ICREA Research Professor at the Institute for Bioengineering of Catalonia (IBEC).

Staff member publications

Gómez-Santacana, Xavier, Pittolo, Silvia, Rovira, Xavier, Lopez, Marc, Zussy, Charleine, Dalton, James A. R., Faucherre, Adèle, Jopling, Chris, Pin, Jean-Philippe, Ciruela, Francisco, Goudet, Cyril, Giraldo, Jesús, Gorostiza, Pau, Llebaria, Amadeu, (2017). Illuminating phenylazopyridines to photoswitch metabotropic glutamate receptors: From the flask to the animals ACS Central Science 3, (1), 81-91

Phenylazopyridines are photoisomerizable compounds with high potential to control biological functions with light. We have obtained a series of phenylazopyridines with light dependent activity as negative allosteric modulators (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5). Here we describe the factors needed to achieve an operational molecular photoisomerization and its effective translation into in vitro and in vivo receptor photoswitching, which includes zebrafish larva motility and the regulation of the antinociceptive effects in mice. The combination of light and some specific phenylazopyridine ligands displays atypical pharmacological profiles, including light-dependent receptor overactivation, which can be observed both in vitro and in vivo. Remarkably, the localized administration of light and a photoswitchable compound in the peripheral tissues of rodents or in the brain amygdalae results in an illumination-dependent analgesic effect. The results reveal a robust translation of the phenylazopyridine photoisomerization to a precise photoregulation of biological activity.


López-Martínez, Montserrat, Artés, Juan Manuel, Sarasso, Veronica, Carminati, Marco, Díez-Pérez, Ismael, Sanz, Fausto, Gorostiza, Pau, (2017). Differential Electrochemical Conductance Imaging at the Nanoscale Small Early View (Online Version of Record published before inclusion in an issue)

Electron transfer in proteins is essential in crucial biological processes. Although the fundamental aspects of biological electron transfer are well characterized, currently there are no experimental tools to determine the atomic-scale electronic pathways in redox proteins, and thus to fully understand their outstanding efficiency and environmental adaptability. This knowledge is also required to design and optimize biomolecular electronic devices. In order to measure the local conductance of an electrode surface immersed in an electrolyte, this study builds upon the current–potential spectroscopic capacity of electrochemical scanning tunneling microscopy, by adding an alternating current modulation technique. With this setup, spatially resolved, differential electrochemical conductance images under bipotentiostatic control are recorded. Differential electrochemical conductance imaging allows visualizing the reversible oxidation of an iron electrode in borate buffer and individual azurin proteins immobilized on atomically flat gold surfaces. In particular, this method reveals submolecular regions with high conductance within the protein. The direct observation of nanoscale conduction pathways in redox proteins and complexes enables important advances in biochemistry and bionanotechnology.

Keywords: Differential electrochemical conductance, ECSTM, Electron transport pathway, Iron passivation, Redox metalloproteins


Gómez-Santacana, Xavier, Dalton, James A. R., Rovira, Xavier, Pin, Jean Philippe, Goudet, Cyril, Gorostiza, Pau, Giraldo, Jesús, Llebaria, Amadeu, (2017). Positional isomers of bispyridine benzene derivatives induce efficacy changes on mGlu5 negative allosteric modulation European Journal of Medicinal Chemistry 127, 567-576

Modulation of metabotropic glutamate receptor 5 (mGlu5) with partial allosteric antagonists has received increased interest due to their favourable in vivo activity profiles compared to the unfavourable side-effects of full inverse agonists. Here we report on a series of bispyridine benzene derivatives with a functional molecular switch affecting antagonistic efficacy, shifting from inverse agonism to partial antagonism with only a single change in the substitution pattern of the benzene ring. These efficacy changes are explained through computational docking, revealing two different receptor conformations of different energetic stability and different positional isomer binding preferences.

Keywords: mGlu5, Isomers, Partial efficacy, NAM, Antagonist, Inverse agonist


Bregestovski, Piotr, Maleeva, Galyna, Gorostiza, Pau, (2017). Light-induced regulation of ligand-gated channel activity British Journal of Pharmacology Epub ahead of print

The control of ligand-gated receptors with light using photochromic compounds has evolved from the first handcrafted examples to accurate, engineered receptors, whose development is supported by rational design, high-resolution protein structures, comparative pharmacology and molecular biology manipulations. Photoswitchable regulators have been designed and characterized for a large number of ligand-gated receptors in the mammalian nervous system, including nicotinic acetylcholine, glutamate and GABA receptors. They provide a well-equipped toolbox to investigate synaptic and neuronal circuits in all-optical experiments. This focused review discusses the design and properties of these photoswitches, their applications and shortcomings and future perspectives in the field.


Terni, Beatrice, Pacciolla, Paolo, Masanas, Helena, Gorostiza, Pau, Llobet, Artur, (2017). Tight temporal coupling between synaptic rewiring of olfactory glomeruli and the emergence of odor-guided behavior in Xenopus tadpoles Journal of Comparative Neurology Early View (Online Version of Record published before inclusion in an issue)

Olfactory sensory neurons (OSNs) are chemoreceptors that establish excitatory synapses within glomeruli of the olfactory bulb. OSNs undergo continuous turnover throughout life, causing the constant replacement of their synaptic contacts. Using Xenopus tadpoles as an experimental system to investigate rewiring of glomerular connectivity, we show that novel OSN synapses can transfer information immediately after formation, mediating olfactory-guided behavior. Tadpoles recover the ability to detect amino acids 4 days after bilateral olfactory nerve transection. Restoration of olfactory-guided behavior depends on the efficient reinsertion of OSNs to the olfactory bulb. Presynaptic terminals of incipient synaptic contacts generate calcium transients in response to odors, triggering long lasting depolarization of olfactory glomeruli. The functionality of reconnected terminals relies on well-defined readily releasable and cytoplasmic vesicle pools. The continuous growth of non-compartmentalized axonal processes provides a vesicle reservoir to nascent release sites, which contrasts to the gradual development of cytoplasmic vesicle pools in conventional excitatory synapses. The immediate availability of fully functional synapses upon formation supports an age-independent contribution of OSNs to the generation of odor maps.

Keywords: Olfactory receptor neurons, Olfactory bulb, Presynaptic terminals, RRID:SCR_013731, RRID:SCR_007164, RRID: AB-887824, RRID: AB-221570, Synaptic vesicles


Rovira, Xavier, Trapero, Ana, Pittolo, Silvia, Zussy, Charleine, Faucherre, Adèle, Jopling, Chris, Giraldo, Jesús, Pin, Jean-Philippe, Gorostiza, Pau, Goudet, Cyril, Llebaria, Amadeu, (2016). OptoGluNAM4.1, a Photoswitchable allosteric antagonist for real-time control of mGlu4 receptor activity Cell Chemical Biology 23, (8), 929-934

OptoGluNAM4.1, a negative allosteric modulator (NAM) of metabotropic glutamate receptor 4 (mGlu4) contains a reactive group that covalently binds to the receptor and a blue-light-activated, fast-relaxing azobenzene group that allows reversible receptor activity photocontrol in vitro and in vivo. OptoGluNAM4.1 induces light-dependent behavior in zebrafish and reverses the activity of the mGlu4 agonist LSP4-2022 in a mice model of chronic pain, defining a photopharmacological tool to better elucidate the physiological roles of the mGlu4 receptor in the nervous system.


Izquierdo-Serra, M., Bautista-Barrufet, A., Trapero, A., Garrido-Charles, A., Diaz-Tahoces, A., Camarero, N., Pittolo, S., Valbuena, S., Perez-Jimenez, A., Gay, M., Garcia-Moll, A., Rodriguez-Escrich, C., Lerma, J., De La Villa, P., Fernandez, E., Pericas, M. A., Llebaria, A., Gorostiza, P., (2016). Optical control of endogenous receptors and cellular excitability using targeted covalent photoswitches Nature Communications 7, 12221

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.


A. R. Dalton, J., Lans, I., Rovira, X., Malhaire, F., Gómez-Santacana, X., Pittolo, S., Gorostiza, P., Llebaria, A., Goudet, C., Pin, J-P., Giraldo, J., (2016). Shining light on an mGlu5 photoswitchable NAM: A theoretical perspective Current Neuropharmacology 14, (5), 441-454

Metabotropic glutamate receptors (mGluRs) are important drug targets because of their involvement in several neurological diseases. Among mGluRs, mGlu5 is a particularly high-profile target because its positive or negative allosteric modulation can potentially treat schizophrenia or anxiety and chronic pain, respectively. Here, we computationally and experimentally probe the functional binding of a novel photoswitchable mGlu5 NAM, termed alloswitch-1, which loses its NAM functionality under violet light. We show alloswitch-1 binds deep in the allosteric pocket in a similar fashion to mavoglurant, the co-crystallized NAM in the mGlu5 transmembrane domain crystal structure. Alloswitch-1, like NAM 2-Methyl-6-(phenylethynyl)pyridine (MPEP), is significantly affected by P655M mutation deep in the allosteric pocket, eradicating its functionality. In MD simulations, we show alloswitch-1 and MPEP stabilize the co-crystallized water molecule located at the bottom of the allosteric site that is seemingly characteristic of the inactive receptor state. Furthermore, both NAMs form H-bonds with S809 on helix 7, which may constitute an important stabilizing interaction for NAM-induced mGlu5 inactivation. Alloswitch-1, through isomerization of its amide group from trans to cis is able to form an additional interaction with N747 on helix 5. This may be an important interaction for amide-containing mGlu5 NAMs, helping to stabilize their binding in a potentially unusual cis-amide state. Simulated conformational switching of alloswitch-1 in silico suggests photoisomerization of its azo group from trans to cis may be possible within the allosteric pocket. However, photoexcited alloswitch-1 binds in an unstable fashion, breaking H-bonds with the protein and destabilizing the co-crystallized water molecule. This suggests photoswitching may have destabilizing effects on mGlu5 binding and functionality.

Keywords: Allosteric modulation, Docking, Metabotropic glutamate receptor, Molecular dynamics, Mutation, Protein structure, Transmembrane domain


Martín-Quirós, Andrés, Nevola, Laura, Eckelt, Kay, Madurga, Sergio, Gorostiza, Pau, Giralt, Ernest, (2015). Absence of a stable secondary structure is not a limitation for photoswitchable inhibitors of Chemistry & Biology 22, (1), 31-37

Many protein-protein interactions (PPIs) are mediated by short, often helical, linear peptides. Molecules mimicking these peptides have been used to inhibit their PPIs. Recently, photoswitchable peptides with little secondary structure have been developed as modulators of clathrin-mediated endocytosis. Here we perform a systematic analysis of a series of azobenzene-crosslinked peptides based on a


Gascón-Moya, Marta, Pejoan, Arnau, Izquierdo-Serra, M., Pittolo, Silvia, Cabrè, Gisela, Hernando, Jordi, Alibés, Ramon, Gorostiza, Pau, Busque, Felix, (2015). An optimized glutamate receptor photoswitch with sensitized azobenzene isomerization Journal of Organic Chemistry 80, (20), 9915-9925

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 non-linear 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. 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 non-linear 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.


Giannotti, Marina I., Cabeza de Vaca, Israel, Artés, Juan Manuel, Sanz, Fausto, Guallar, Victor, Gorostiza, Pau, (2015). Direct measurement of the nanomechanical stability of a redox protein active site and its dependence upon metal binding Journal of Physical Chemistry B 119, (36), 12050-12058

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. 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.


Eckelt, Kay, Masanas, Helena, Llobet, Artur, Gorostiza, P., (2014). Automated high-throughput measurement of body movements and cardiac activity of Xenopus tropicalis tadpoles Journal of Biological Methods 1, (2), e9

Xenopus tadpoles are an emerging model for developmental, genetic and behavioral studies. A small size, optical accessibility of most of their organs, together with a close genetic and structural relationship to humans make them a convenient experimental model. However, there is only a limited toolset available to measure behavior and organ function of these animals at medium or high-throughput. Herein, we describe an imaging-based platform to quantify body and autonomic movements of Xenopus tropicalis tadpoles of advanced developmental stages. Animals alternate periods of quiescence and locomotor movements and display buccal pumping for oxygen uptake from water and rhythmic cardiac movements. We imaged up to 24 animals in parallel and automatically tracked and quantified their movements by using image analysis software. Animal trajectories, moved distances, activity time, buccal pumping rates and heart beat rates were calculated and used to characterize the effects of test compounds. We evaluated the effects of propranolol and atropine, observing a dose-dependent bradycardia and tachycardia, respectively. This imaging and analysis platform is a simple, cost-effective high-throughput in vivo assay system for genetic, toxicological or pharmacological characterizations.

Keywords: Xenopus tropicalis, Animal behavior, Cardiac imaging, Motion analysis, Animal tracking, Hhigh-throughput in vivo assay


Pittolo, Silvia, Gómez-Santacana, Xavier, Eckelt, Kay, Rovira, Xavier, Dalton, James, Goudet, Cyril, Pin, Jean-Philippe, Llobet, Artur, Giraldo, Jesús, Llebaria, Amadeu, Gorostiza, Pau, (2014). An allosteric modulator to control endogenous G protein-coupled receptors with light Nature Chemical Biology 10, (10), 813-815

Controlling drug activity with light offers the possibility of enhancing pharmacological selectivity with spatial and temporal regulation, thus enabling highly localized therapeutic effects and precise dosing patterns. Here we report on the development and characterization of what is to our knowledge the first photoswitchable allosteric modulator of a G protein–coupled receptor. Alloswitch-1 is selective for the metabotropic glutamate receptor ​mGlu5 and enables the optical control of endogenous ​mGlu5 receptors.


Izquierdo-Serra, M., Gascón-Moya, Marta, Hirtz, Jan J., Pittolo, Silvia, Poskanzer, Kira E., Ferrer, Eric, Alibés, Ramon, Busque, Felix, Yuste, Rafael, Hernando, Jordi, Gorostiza, Pau, (2014). Two-photon neuronal and astrocytic stimulation of azobenzene-based photoswitches Journal of the American Chemical Society American Chemical Society 136, (24), 8693-8701

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 has 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.


Bautista-Barrufet, A., López-Gallego, F., Rojas-Cervellera, V., Rovira, C., Pericàs, M. A., Guisán, J. M., Gorostiza, P., (2014). Optical control of enzyme enantioselectivity in solid phase ACS Catalysis 4, (3), 1004-1009

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. 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.


Artés, Juan M., López-Martínez, Montserrat, Díez-Pérez, Ismael, Sanz, Fausto, Gorostiza, Pau, (2014). Conductance switching in single wired redox proteins Small 10, (13), 2537-2541

Switching events in the current flowing through individual redox proteins, (azurin) spontaneously wired between two electrodes, are studied using an electrochemical scanning tunneling microscope (ECSTM). These switching events in the current–time trace are characterized using conductance histograms, and reflect the intrinsic redox thermodynamic dispersion in the azurin population. This conductance switching may pose limitations to miniaturizing redox protein-based devices.

Keywords: Bioelectronics, Protein transistors, Molecular junctions, Switches, STM


Lagunas, A., Garcia, A., Artés, J. M., Vida, Y., Collado, D., Pérez-Inestrosa, E., Gorostiza, P., Claros, S., Andrades, J. A., Samitier, J., (2014). Large-scale dendrimer-based uneven nanopatterns for the study of local arginine-glycine-aspartic acid (RGD) density effects on cell adhesion Nano Research 7, (3), 399-409

Cell adhesion processes are governed by the nanoscale arrangement of the extracellular matrix (ECM), being more affected by local rather than global concentrations of cell adhesive ligands. In many cell-based studies, grafting of dendrimers on surfaces has shown the benefits of the local increase in concentration provided by the dendritic configuration, although the lack of any reported surface characterization has limited any direct correlation between dendrimer disposition and cell response. In order to establish a proper correlation, some control over dendrimer surface deposition is desirable. Here, dendrimer nanopatterning has been employed to address arginine-glycine-aspartic acid (RGD) density effects on cell adhesion. Nanopatterned surfaces were fully characterized by atomic force microscopy (AFM), scanning tunneling microscopy (STM) and X-ray photoelectron spectroscopy (XPS), showing that tunable distributions of cell adhesive ligands on the surface are obtained as a function of the initial dendrimer bulk concentration. Cell experiments showed a clear correlation with dendrimer surface layout: Substrates presenting regions of high local ligand density resulted in a higher percentage of adhered cells and a higher degree of maturation of focal adhesions (FAs). Therefore, dendrimer nanopatterning is presented as a suitable and controlled approach to address the effect of local ligand density on cell response. Moreover, due to the easy modification of dendrimer peripheral groups, dendrimer nanopatterning can be further extended to other ECM ligands having density effects on cells.

Keywords: Arginine-glycine-aspartic acid, Atomic force microscopy, Cell adhesion, Dendrimer, Focal adhesions, Scanning tunneling microscopy


Bahamonde, María Isabel, Taura, Jaume, Paoletta, Silvia, Gakh, Andrei Alexandrovich, Chakraborty, Saibal, Hernando, Jordi, Fernández-Dueñas, Víctor, Jacobson, Kenneth A., Gorostiza, Pau, Ciruela, Francisco, (2014). Photomodulation of G protein-coupled adenosine receptors by a novel light-switchable ligand Bioconjugate Chemistry American Chemical Society 25, (10), 1847-1854

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 non-selective adenosine receptor agonist, nucleoside derivative MRS5543 containing an aryl diazo linkage on the N6 substituent, which in the dark (relaxed isomer) behaved as a full adenosine A3 receptor (A3R) and partial adenosine A2A receptor (A2AR) agonist. Conversely, upon photoisomerization with blue light (460 nm), it remained a full A3R agonist but became an A2AR 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. 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 non-selective adenosine receptor agonist, nucleoside derivative MRS5543 containing an aryl diazo linkage on the N6 substituent, which in the dark (relaxed isomer) behaved as a full adenosine A3 receptor (A3R) and partial adenosine A2A receptor (A2AR) agonist. Conversely, upon photoisomerization with blue light (460 nm), it remained a full A3R agonist but became an A2AR 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.


Artés, J. M., López-Martínez, M., Díez-Pérez, I., Sanz, F., Gorostiza, P., (2014). Nanoscale charge transfer in redox proteins and DNA: Towards biomolecular electronics Electrochimica Acta 140, 83-95

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.

Keywords: Bioelectrochemistry, Biomolecular electronics, Charge transfer, Nanobiodevice, Single-molecule junction


Gomez-Santacana, X., Rovira, X., Dalton, J. A., Goudet, C., Pin, J. P., Gorostiza, P., Giraldo, J., Llebaria, A., (2014). A double effect molecular switch leads to a novel potent negative allosteric modulator of metabotropic glutamate receptor 5 MedChemComm 5, (10), 1548-1554

Compounds that modulate the function of G-protein-coupled receptors (GPCRs) by binding to their allosteric sites are of potential interest for the treatment of multiple CNS and non-CNS disorders. Allosteric ligands can act either as positive (PAM), negative (NAM), or silent (SAM) receptor modulators and have numerous advantages over classic orthosteric compounds, including improved GPCR-subtype selectivity; the capacity to adapt to physiological conditions; and better safety profiles. Despite these benefits, allosteric modulators are difficult to design and optimize and are often prone to "molecular switching": a structural phenomenon by which very subtle chemical variations in the ligand result in unexpected changes in selectivity profiles or pharmacology, changing PAMs to NAMs or vice versa. Here, we report the discovery of a nanomolar and subtype selective NAM of metabotropic glutamate receptor 5 (mGlu5) through a targeted "double effect molecular switch" of a potent mGlu4 PAM, and suggests a promising approach towards the discovery of novel mGluR allosteric modulators.


Bautista-Barrufet, Antoni, Izquierdo-Serra, M., Gorostiza, Pau, (2014). Photoswitchable Ion Channels and Receptors Advances in Atom and Single Molecule Machines Novel Approaches for Single Molecule Activation and Detection (ed. Benfenati, Fabio, Di Fabrizio, Enzo, Torre, Vincent), Springer Berlin Heidelberg , 169-188

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.


Gorostiza, Pau, Arosio, Daniele, Bregestovski, Piotr, (2013). Molecular probes and switches for functional analysis of receptors, ion channels and synaptic networks Frontiers in Molecular Neuroscience 6, (Article 48), 1-2

Izquierdo-Serra, Mercè, Trauner, Dirk, Llobet, Artur, Gorostiza, Pau, (2013). Optical modulation of neurotransmission using calcium photocurrents through the ion channel LiGluR Frontiers in Molecular Neuroscience 6, (Article 3), 1-6

A wide range of light-activated molecules (photoswitches and phototriggers) have been used to the study of computational properties of an isolated neuron by acting pre and postsynaptically. However, new tools are being pursued to elicit a presynaptic calcium influx that triggers the release of neurotransmitters, most of them based in calcium-permeable Channelrhodopsin-2 mutants. Here we describe a method to control exocytosis of synaptic vesicles through the use of a light-gated glutamate receptor (LiGluR), which has recently been demonstrated that supports secretion by means of calcium influx in chromaffin cells. Expression of LiGluR in hippocampal neurons enables reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the wavelength of illumination. This method may be useful for the determination of the complex transfer function of individual synapses.

Keywords: Calcium, Neurotransmission, Optogenetics, Neural coding, Firing rate, Optical control, Synaptic transfer function


Nevola, L., Martín-Quirós, A., Eckelt, K., Camarero, N., Tosi, S., Llobet, A., Giralt, E., Gorostiza, P., (2013). Light-regulated stapled peptides to inhibit protein-protein interactions involved in clathrin-mediated endocytosis Angewandte Chemie - International Edition 52, (30), 7704-7708

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.

Keywords: Clathrin-mediated endocytosis, Optopharmacology, Peptides, Photoswitches, Protein-protein interactions


Izquierdo-Serra, Mercè, Trauner, Dirk, Llobet, Artur, Gorostiza, Pau, (2013). Optical control of calcium-regulated exocytosis Biochimica et Biophysica Acta (BBA) - General Subjects 1830, (3), 2853-2860

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 and general significance 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. Thus, 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.

Keywords: Optical control, Calcium, Exocytosis, Light-gated glutamate receptor (LiGluR), Neurotransmission, Optogenetics


Raster, P., Späth, A., Bultakova, S., Gorostiza, P., König, B., Bregestovski, P., (2013). New GABA amides activating GABAA-receptors Beilstein Journal of Organic Chemistry 9, 406-410

We have prepared a series of new and some literature-reported GABA-amides and determined their effect on the activation of GABA A-receptors expressed in CHO cells. Special attention was paid to the purification of the target compounds to remove even traces of GABA contaminations, which may arise from deprotection steps in the synthesis. GABA-amides were previously reported to be partial, full or superagonists. In our hands these compounds were not able to activate GABAA-receptor channels in whole-cell patch-clamp recordings. New GABA-amides, however, gave moderate activation responses with a clear structure-activity relationship suggesting some of these compounds as promising molecular tools for the functional analysis of GABAA-receptors.


Gorostiza, Pau, (2012). Control celular mediante luz Investigación y Ciencia 433, 11-12

Artés, Juan M., Díez-Pérez, Ismael, Gorostiza, Pau, (2012). Transistor-like behavior of single metalloprotein junctions Nano Letters 12, (6), 2679-2684

Single protein junctions consisting of azurin bridged between a gold substrate and the probe of an electrochemical tunneling microscope (ECSTM) have been obtained by two independent methods that allowed statistical analysis over a large number of measured junctions. Conductance measurements yield (7.3 ± 1.5) ? 10–6G0 in agreement with reported estimates using other techniques. Redox gating of the protein with an on/off ratio of 20 was demonstrated and constitutes a proof-of-principle of a single redox protein field-effect transistor.


Artés, Juan M., López-Martínez, Montserrat, Giraudet, Arnaud, Díez-Pérez, Ismael, Sanz, Fausto, Gorostiza, Pau, (2012). Current–Voltage characteristics and transition voltage spectroscopy of individual redox proteins Journal of the American Chemical Society 134, (50), 20218-20221

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. 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.


Simao, C., Mas-Torrent, M., Crivillers, N., Lloveras, V., Artés, Juan Manuel, Gorostiza, Pau, Veciana, Jaume, Rovira, C., (2011). A robust molecular platform for non-volatile memory devices with optical and magnetic responses Nature Chemistry 3, (5), 359-364

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.

Keywords: Self-assembled monolayers, Chromophore-based monolayers, Ultrathin platinum films, Carbon free-radicals, Per-million levels, Polychlorotriphenylmethyl radicals, Electron-transfer, Surface, Logic, Quantification


Artés, Juan M., Díez-Pérez, Ismael, Sanz, Fausto, Gorostiza, Pau, (2011). Direct measurement of electron transfer distance decay constants of single redox proteins by electrochemical tunneling spectroscopy ACS Nano 5, (3), 2060-2066

We present a method to measure directly and at the single-molecule level the distance decay constant that characterizes the rate of electron transfer (ET) in redox proteins. Using an electrochemical tunneling microscope under bipotentiostatic control, we obtained current-distance spectroscopic recordings of individual redox proteins confined within a nanometric tunneling gap at a well-defined molecular orientation. The tunneling current decays exponentially, and the corresponding decay constant (

Keywords: Long-range electron transfer (LRET), Distance decay constant, Single-molecule electrochemistry, Redox enzyme, Metalloprotein, Blue copper protein, Azurin, Electrochemical scanning tunneling microscopy and spectroscopy, Nanoelectrodes, Debye length, Electrochemical charge screening


Gorostiza, P., Isacoff, E.Y., (2011). Photoswitchable ligand-gated ion channels Photosensitive molecules for controlling biological function (ed. Chambers, J. J. , Kramer, R. H.), Springer (Saskatoon, Canada) 55, 267-285

Ligand-activated proteins can be controlled with light by means of synthetic photoisomerizable tethered ligands (PTLs). The application of PTLs to ligand-gated ion channels, including the nicotinic acetylcholine receptor and ionotropic glutamate receptors, is reviewed with emphasis on rational photoswitch design and the mechanisms of optical switching. Recently reported molecular dynamic methods allow simulation with high reliability of novel PTLs for any ligand-activated protein whose structure is known.

Keywords: Nicotinic acetylcholine receptor, Kainate receptor, Glutamate receptor, Photoisomerizable tether ligand (PTL), Optical switch, Nanotoggle, Azobenzene, Neurobiology,, Nanoengineering, Nanomedicine


Caballero-Briones, F., Artes, J. M., Diez-Perez, I., Gorostiza, P., Sanz, F., (2009). Direct observation of the valence band edge by in situ ECSTM-ECTS in p-type Cu2O layers prepared by copper anodization Journal of Physical Chemistry C 113, (3), 1028-1036

Polycrystalline Cu2O layers have been selectively grown by electrochemical anodization of polycrystalline Cu electrodes in an alkaline medium (pH 12.85). Uniform layers with thicknesses around 100 nm have been obtained. Using electrochemical impedance spectroscopy, it was concluded that the Cu2O films behave as a p-type semiconductor. The Mott-Schottky plot gives a value for the flat band potential of U-FB = -255 mV vs silver/silver chloride electrode (SSC), an estimated carrier density N-A = 6.1 x 10(17) cm(-3), and the space charge layer width was calculated to be W-SCL = 9 nm at a band bending of 120 mV. The electronic structure of the Cu vertical bar Cu2O vertical bar electrolyte interface was for the first time probed by in situ electrochemical tunneling spectroscopy. The use of in situ electrochemical scanning tunneling microscopy allows us to directly observed the valence band edge and determine its position against the absolute energy scale to be E-VB = -4.9 eV. Finally, we constructed a quantitative electronic diagram of the Cu vertical bar Cu2O vertical bar electrolyte interface, where the positions of the valence and conduction band edges are depicted, as well as the edge of the previously reported electronic subband.

Keywords: 0.1 m NaOH, Electrochemical tunneling spectroscopy, Cuprous-oxide films, Anodic-oxidation, Electronic-structure, Alkaline-solution, Aqueous-solution, Initial-stages, Passive film, Thin-films


Gorostiza, P., Isacoff, E. Y., (2008). Optical switches for remote and noninvasive control of cell signaling Science 322, (5900), 395-399

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.

Keywords: Ion channels, Acetylcholine receptor, Glutamate-receptor, Potassium channel, K+ channel, Light, Neurons, Channelrhodopsin-2, Manipulation, Activation


Gorostiza, P., Isacoff, E. Y., (2008). Nanoengineering ion channels for optical control Physiology 23, (5), 238-247

Chemical modification with photoisomerizable tethered ligands endows proteins with sensitivity to light. These optically actuated proteins are revolutionizing research in biology by making it possible to manipulate biological processes noninvasively and with unprecedented spatiotemporal resolution.

Keywords: -----


Díez-Pérez, Ismael, Guell, Aleix Garcia, Sanz, Fausto, Gorostiza, Pau, (2006). Conductance maps by electrochemical tunneling spectroscopy to fingerprint the electrode electronic structure Analytical Chemistry 78, (20), 7325-7329

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.

Keywords: Passive film, Oxide-film, Stainless-steel, Iron, Microscope, Surfaces, STM, Probes


Díez-Pérez, Ismael, Sanz, Fausto, Gorostiza, Pau, (2006). Electronic barriers in the iron oxide film govern its passivity and redox behavior: Effect of electrode potential and solution pH Electrochemistry Communications 8, (10), 1595-1602

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


Díez-Pérez, Ismael, Vericat, Carolina, Gorostiza, Pau, Sanz, Fausto, (2006). The iron passive film breakdown in chloride media may be mediated by transient chloride-induced surface states located within the band gap Electrochemistry Communications 8, (4), 627-632

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 (~3.3eV). 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


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