Reversibly caged glutamate: a photochromic agonist of ionotropic glutamate receptors.
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Action Potentials↗
Biomedical subjects
Pau Gorostiza
Publications and source records attributed to Pau Gorostiza.
The twisted ion-permeation pathway of a resting voltage-sensing domain.
Proteins containing voltage-sensing domains (VSDs) translate changes in membrane potential into changes in ion permeability or enzymatic activity. In channels, voltage change triggers a switch in conformation of the VSD, which drives gating in a separate pore domain, or, in channels lacking a pore domain, directly gates an ion pathway within the VSD. Neither mechanism is well understood. In the Shaker potassium channel, mutation of the first arginine residue of the S4 helix to a smaller uncharged residue makes the VSD permeable to ions ('omega current') in the resting conformation ('S4 down'). Here we perform a structure-guided perturbation analysis of the omega conductance to map its VSD permeation pathway. We find that there are four omega pores per channel, which is consistent with one conduction path per VSD. Permeating ions from the extracellular medium enter the VSD at its peripheral junction with the pore domain, and then plunge into the core of the VSD in a curved conduction pathway. Our results provide a model of the resting conformation of the VSD.
Animals↗
Conductance maps by electrochemical tunneling spectroscopy to fingerprint the electrode electronic structure.
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.
Electrochemistry↗
Titration force microscopy on supported lipid bilayers.
The use of chemically modified atomic force microscopy (AFM) probes allows us to measure the surface charges of supported planar lipid bilayers with high sensitivity through the force spectroscopy operation mode. By controlling the chemistry of the tip, we can perform a classical analytical chemistry titration where the titration agent is a weak acid (attached to the AFM tip) with the particularity of being performed in surface rather than in solution and, especially, at the nanometric scale. Thus, the AFM tip acts as a real "nanosensor". The approaching curves of the force plots reveal that electrostatic interactions between the tip and the supported membrane play a key role. Besides, the plot of the adhesion force (measured from the retracting curve of the force plots) versus pH displays a nonsigmoidal shape with a peak in the adhesion force attributed to high-energy hydrogen bonds. One of these peaks corresponds to the pKa of the surface under study and the other to the pKa of the titrating probe attached to the tip.
Dimyristoylphosphatidylcholine↗
Allosteric control of an ionotropic glutamate receptor with an optical switch.
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.
Allosteric Regulation↗
Nanomechanics of silicon surfaces with atomic force microscopy: an insight to the first stages of plastic deformation.
The use of stiff cantilevers with diamond tips allows us to perform nanoindentations on hard covalent materials such as silicon with atomic force microscopy. Thanks to the high sensitivity in the force measurements together with the high resolution upon imaging the surface, we can study nanomechanical properties. At this scale, the surface deforms, following a simple non-Hertzian spring model. The plastic onset can be assessed from a discontinuity in the force-distance curves. Hardness measurements with penetration depths as small as 1 nm yield H= approximately 25 GPa, thus showing a drastic increase with penetration depths below 5 nm.
Computer Simulation↗
Molecular handles for the mechanical manipulation of single-membrane proteins in living cells.
We have developed a procedure to selectively biotinylate a specific membrane protein, enabling its attachment to external force probes and thus allowing its mechanical manipulation within its native environment. Using potassium channels as model membrane proteins in oocytes, we have found that Maleimide-PEG3400-biotin is the crosslinker with highest conjugation selectivity and accessibility to external probes. Neutravidin-coated beads provide for directed attachment while avoiding nonspecific interactions with the cell. The technology was successfully tested by mechanical manipulation of biotinylated extracellular residues of channels in oocytes using an atomic force microscope under conditions which preserve function of the channels. Binding forces of approximately 80 pN at 100 nN/s were measured.
Animals↗
Preparation of reliable probes for electrochemical tunneling spectroscopy.
We present a new procedure to prepare Pt/Ir probes for electrochemical scanning tunneling microscopy (STM) and spectroscopy applications. We detail the experimental setup and the improvements over previous methods. The probes have been used successfully for measurements of tunneling spectroscopy under electrochemical control, which requires scanning the potential of the tip at high velocity.
Journal Article↗
Collective behaviour in two-dimensional cobalt nanoparticle assemblies observed by magnetic force microscopy.
The use of magnetic nanoparticles in the development of ultra-high-density recording media is the subject of intense research. Much of the attention of this research is devoted to the stability of magnetic moments, often neglecting the influence of dipolar interactions. Here, we explore the magnetic microstructure of different assemblies of monodisperse cobalt single-domain nanoparticles by magnetic force microscopy and magnetometric measurements. We observe that when the density of particles per unit area is higher than a determined threshold, the two-dimensional self-assemblies behave as a continuous ferromagnetic thin film. Correlated areas (similar to domains) of parallel magnetization roughly ten particles in diameter appear. As this magnetic percolation is mediated by dipolar interactions, the magnetic microstructure, its distribution and stability, is strongly dependent on the topological distribution of the dipoles. Thus, the magnetic structures of three-dimensional assemblies are magnetically soft, and an evolution of the magnetic microstructure is observed with consecutive scans of the microscope tip.
Anisotropy↗
Alkali halide nanocrystal growth and etching studied by AFM and modeled by MD simulations.
NaCl hillocks have been grown on the NaCl(100) monocrystalline surface by simply making the tip of an atomic force microscope cantilever interact with the surface. A quantitative discussion about the hillock dissolution process as well as physical stability of these ionic surfaces has been made. Molecular dynamics simulations helped us to interpret the experimental data. An explanation for the hillock formation and dissolution phenomena is also discussed.
Journal Article↗
Control of neurotransmitter release by an internal gel matrix in synaptic vesicles.
Neurotransmitters are stored in synaptic vesicles, where they have been assumed to be in free solution. Here we report that in Torpedo synaptic vesicles, only 5% of the total acetylcholine (ACh) or ATP content is free, and that the rest is adsorbed to an intravesicular proteoglycan matrix. This matrix, which controls ACh and ATP release by an ion-exchange mechanism, behaves like a smart gel. That is, it releases neurotransmitter and changes its volume when challenged with small ionic concentration change. Immunodetection analysis revealed that the synaptic vesicle proteoglycan SV2 is the core of the intravesicular matrix and is responsible for immobilization and release of ACh and ATP. We suggest that in the early steps of vesicle fusion, this internal matrix regulates the availability of free diffusible ACh and ATP, and thus serves to modulate the quantity of transmitter released.
Acetylcholine↗
Self-assembly of drug-polymer complexes: a spontaneous nanoencapsulation process monitored by atomic force microscopy.
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.
Carrageenan↗
Supramolecular properties of the proline-rich gamma-Zein N-terminal domain.
Zeins are maize storages proteins that accumulate inside large vesicles called protein bodies. gamma-Zein lines the inner face of the protein body membrane, and its N-terminal proline-rich repetitive domain with the sequence (VHLPPP)(8) appears to be necessary for the accumulation of the protein within the organelle. Synthetic (VHLPPP)(8) adopts an amphipathic polyproline II conformation. In a preliminary recent work we used atomic force microscopy to study the surface organization of the octamer and transmission electron microscopy to visualize aggregates of the peptide from aqueous solution. We previously envisioned two self-assembly models (i.e., the geometric and the micellar) that take into account the observed features. In the present work we studied in detail the self-assembly of the peptide in solution and found that the peptide is able to form cylindrical micelles. Fibrils formed on graphite are generated by assembly of solution micelles. Based on the results of these studies, we focused exclusively on the micellar model. To our knowledge we have characterized for the first time supramolecular aggregates of polyproline structures other than collagen. The spontaneous arrangement of (VHLPPP)(8) suggests a role for the N-terminal domain of gamma-zein in the process of the whole protein deposition in protein bodies.
Biophysical Phenomena↗