Publications by authors named "Arin Marchesi"

Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation.

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Laminins are trimeric glycoproteins with important roles in cell-matrix adhesion and tissue organization. The laminin α, ß, and γ-chains have short N-terminal arms, while their C-termini are connected via a triple coiled-coil domain, giving the laminin molecule a well-characterized cross-shaped morphology as a result. The C-terminus of laminin alpha chains contains additional globular laminin G-like (LG) domains with important roles in mediating cell adhesion.

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Article Synopsis
  • Transmembrane protein 16F (TMEM16F) is a calcium-activated protein that works as both an ion channel and a phospholipid scramblase, showing diverse structural forms that impact its function.
  • Using atomic force microscopy, researchers observed various TMEM16F assemblies that have not been seen in previous high-resolution studies, revealing different dimerization and protomer orientations.
  • The study establishes a link between calcium-induced activation and structural changes in TMEM16F, suggesting that its conformational diversity plays a crucial role in its physiological activities, particularly in lipid and ion transport.
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  • Programmable DNA binding and cleavage using CRISPR-Cas9 has transformed life sciences, but off-target effects limit its broader application in biology and medicine.
  • Understanding how Cas9 interacts with DNA is essential for enhancing genome editing efficiency; this study investigates these dynamics using high-speed atomic force microscopy (HS-AFM).
  • The findings reveal that SaCas9 forms a flexible bilobed structure when bound to sgRNA, engages in attractive interactions with target DNA, and follows a unique process of local DNA bending that leads to stable complex formation before cleavage.
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Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscopy (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer.

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Atomic force microscopy (AFM) can visualize the dynamics of single biomolecules under near-physiological conditions. However, the scanning tip probes only the molecular surface with limited resolution, missing details required to fully deduce functional mechanisms from imaging alone. To overcome such drawbacks, we developed a computational framework to reconstruct 3D atomistic structures from AFM surface scans, employing simulation AFM and automatized fitting to experimental images.

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Fast and selective recognition of molecules at the nanometer scale without labeling is a much desired but still challenging goal to achieve. Here, we show the use of high-speed atomic force microscopy (HS-AFM) for real-time and real-space recognition of unlabeled membrane receptors using tips conjugated with small synthetic macrocyclic peptides. The single-molecule recognition method is validated by experiments on the human hepatocyte growth factor receptor (hMET), which selectively binds to the macrocyclic peptide aMD4.

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Cyclic nucleotide-gated (CNG) channels are key to the signal transduction machinery of certain sensory modalities both in vertebrate and invertebrate organisms. They translate a chemical change in cyclic nucleotide concentration into an electrical signal that can spread through sensory cells. Despite CNG and voltage-gated potassium channels sharing a remarkable amino acid sequence homology and basic architectural plan, their functional properties are dramatically different.

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High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualizing the dynamics of individual biomolecules. However, in single-molecule HS-AFM imaging applications, x,y-scanner ranges are typically restricted to a few hundred nanometers, preventing overview observation of larger molecular assemblies, such as 2-dimensional protein crystal growth or fibrillar aggregation. Previous advances in scanner design using mechanical amplification of the piezo-driven x,y-positioning system have extended the size of HS-AFM image frames to several tens of micrometer, but these large scanners may suffer from mechanical instabilities at high scan speeds and only record images with limited pixel numbers and comparatively low lateral resolutions (> 20-100 nm/pixel), complicating single-molecule analysis.

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Cyclic nucleotide-gated (CNG) ion channels are non-selective cation channels key to signal transduction. The free energy difference of cyclic-nucleotide (cAMP/cGMP) binding/unbinding is translated into mechanical work to modulate the open/closed probability of the pore, i.e.

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ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions.

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Several channels, ranging from TRP receptors to Gap junctions, allow the exchange of small organic solute across cell membrane. However, very little is known about the molecular mechanism of their permeation. Cyclic Nucleotide Gated (CNG) channels, despite their homology with K+ channels and in contrast with them, allow the passage of larger methylated and ethylated ammonium ions like dimethylammonium (DMA) and ethylammonium (EA).

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Single-molecule force spectroscopy (SMFS) measurements allow for quantification of the molecular forces required to unfold individual protein domains. Atomic force microscopy (AFM) is one of the long-established techniques for force spectroscopy (FS). Although FS at conventional AFM pulling rates provides valuable information on protein unfolding, in order to get a more complete picture of the mechanism, explore new regimes, and combine and compare experiments with simulations, we need higher pulling rates and μs-time resolution, now accessible via high-speed force spectroscopy (HS-FS).

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The advent of high-speed atomic force microscopy (HS-AFM) over the recent years has opened up new horizons for the study of structure, function and dynamics of biological molecules. HS-AFM is capable of 1000 times faster imaging than conventional AFM. This circumstance uniquely enables the observation of the dynamics of all the molecules present in the imaging area.

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Mechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using atomic force microscopy on proteins presenting multiple domains have revealed that unfolding forces depend on the number of folded domains (history) and have reported intermediate states and rare events. However, the common use of unspecific attachment approaches to pull the protein of interest holds important limitations to study unfolding history and may lead to discarding rare and multiple probing events due to the presence of unspecific adhesion and uncertainty on the pulling site.

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Cyclic nucleotide-gated (CNG) channels mediate transduction in several sensory neurons. These channels use the free energy of CNs' binding to open the pore, a process referred to as gating. CNG channels belong to the superfamily of voltage-gated channels, where the motion of the α-helix S6 controls gating in most of its members.

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The determination at atomic resolution of the three-dimensional molecular structure of membrane proteins such as receptors and several ion channels has been a major breakthrough in structural biology. The molecular structure of several members of the superfamily of voltage-gated ionic channels such as K and Na is now available. However, despite several attempts, the molecular structure at atomic resolution of the full cyclic nucleotide-gated (CNG) ion channel, although a member of the same superfamily of voltage-gated ion channels, has not been obtained yet, neither by X-ray crystallography nor by electron cryomicroscopy (cryo-EM).

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Article Synopsis
  • CNG ion channels are similar to K(+) channels but allow various monovalent cations to pass through without discrimination, showing permeability to some organic cations as well.
  • Research using electrophysiology, molecular dynamics simulations, and X-ray crystallography reveals that CNG channel pores are highly flexible, changing size when different cations are present.
  • The movement and conformations of specific amino acids in the channel, influenced by the type of ion and voltage, explain the channels' ability to link gating mechanisms with ionic permeation and their lack of selectivity.
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Key Points: Desensitization and inactivation provide a form of short-term memory controlling the firing patterns of excitable cells and adaptation in sensory systems. Unlike many of their cousin K(+) channels, cyclic nucleotide-gated (CNG) channels are thought not to desensitize or inactivate. Here we report that CNG channels do inactivate and that inactivation is controlled by extracellular protons.

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In cyclic nucleotide-gated (CNGA1) channels, in the presence of symmetrical ionic conditions, current-voltage (I-V) relationship depends, in a complex way, on the radius of permeating ion. It has been suggested that both the pore and S4 helix contribute to the observed rectification. In the present manuscript, using tail and gating current measurements from homotetrameric CNGA1 channels expressed in Xenopus oocytes, we clarify and quantify the role of the pore and of the S4 helix.

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Cyclic nucleotide-gated (CNG) channels and K+ channels have a significant sequence identity and are thought to share a similar 3D structure. K+ channels can accommodate simultaneously two or three permeating ions inside their pore and therefore are referred to as multi-ion channels. Also CNGA1 channels are multi-ion channels, as they exhibit an anomalous mole fraction effect (AMFE) in the presence of mixtures of 110 mM Li+ and Cs+ on the cytoplasmic side of the membrane.

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  • * Research shows that while these channels are voltage-independent with ions like Li(+), Na(+), and K(+), they become voltage-dependent with other ions such as Rb(+) and Cs(+).
  • * The study suggests that the evolution of these channels involves a loss of voltage sensitivity when certain ions permeate, which is crucial for the function of photoreceptor cells in vertebrates.
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The aminoacid sequences of CNG and K(+) channels share a significant sequence identity, and it has been suggested that these channels have a common ancestral 3D architecture. However, K(+) and CNG channels have profoundly different physiological properties: indeed, K(+) channels have a high ionic selectivity, their gating strongly depends on membrane voltage and when opened by a steady depolarizing voltage several K(+) channels inactivate, whereas CNG channels have a low ion selectivity, their gating is poorly voltage dependent, and they do not desensitize in the presence of a steady concentration of cyclic nucleotides that cause their opening. The purpose of the present review is to summarize and recapitulate functional and structural differences between K(+) and CNG channels with the aim to understand the gating mechanisms of CNG channels.

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