Two-state Behavior Research Articles

Secondary structure determines electron transport in peptides

Proteins play a key role in biological electron transport, but the structure-function relationships governing the electronic properties of peptides are not fully understood. Despite recent progress, understanding the link between peptide conformational flexibility, hierarchical structures, and electron transport pathways has been challenging. Here, we use single-molecule experiments, molecular dynamics (MD) simulations, nonequilibrium Green's function-density functional theory (NEGF-DFT), and unsupervised machine learning to understand the role of secondary structure on electron transport in peptides. Our results reveal a two-state molecular conductance behavior for peptides across several different amino acid sequences. MD simulations and Gaussian mixture modeling are used to show that this two-state molecular conductance behavior arises due to the conformational flexibility of peptide backbones, with a high-conductance state arising due to a more defined secondary structure (beta turn or 310 helices) and a low-conductance state occurring for extended peptide structures. These results highlight the importance of helical conformations on electron transport in peptides. Conformer selection for the peptide structures is rationalized using principal component analysis of intramolecular hydrogen bonding distances along peptide backbones. Molecular conformations from MD simulations are used to model charge transport in NEGF-DFT calculations, and the results are in reasonable qualitative agreement with experiments. Projected density of states calculations and molecular orbital visualizations are further used to understand the role of amino acid side chains on transport. Overall, our results show that secondary structure plays a key role in electron transport in peptides, which provides broad avenues for understanding the electronic properties of proteins.

A water structure indicator suitable for generic contexts: Two-liquid behavior at hydration and nanoconfinement conditions and a molecular approach to hydrophobicity and wetting.

In a recent work, we have briefly introduced a new structural index for water that, unlike previous indicators, was devised specifically for generic contexts beyond bulk conditions, making it suitable for hydration and nanoconfinement settings. In this work, we shall study this metric in detail, demonstrating its ability to reveal the existence of a fine-tuned interplay between the local structure and energetics in liquid water. This molecular principle enables the establishment of an extended hydrogen bond network, while simultaneously allowing for the existence of network defects by compensating for uncoordinated sites. By studying different water models and different temperatures encompassing both the normal liquid and the supercooled regime, this molecular mechanism will be shown to underlie the two-state behavior of bulk water. In addition, by studying functionalized self-assembled monolayers and diverse graphene-like surfaces, we shall show that this principle is also operative at hydration and nanoconfinement conditions, thus generalizing the validity of the two-liquid scenario of water to these contexts. This approach will allow us to define conditions for wettability, providing an accurate measure of hydrophobicity and a reliable predictor of filling and drying transitions. Hence, it might open the possibility of elucidating the active role of water in the broad fields of biophysics and materials science. As a preliminary step, we shall study the hydration structure and hydrophilicity of graphene-like systems (parallel graphene sheets and carbon nanotubes) as a function of the confinement dimensionality.

Atomistic simulations of RNA duplex thermal denaturation: Sequence- and forcefield-dependence

Double-stranded RNA is the end-product of template-based replication, and is also the functional state of some biological RNAs. Similarly to proteins and DNA, they can be denatured by temperature, with important physiological and technological implications. Here, we use an in silico strategy to probe the thermal denaturation of RNA duplexes. Following previous results that were obtained on a few different duplexes, and which nuanced the canonical 2-state picture of nucleic acid denaturation, we here specifically address three different aspects that greatly improve our description of the temperature-induced dsRNA separation. First, we investigate the effect of the spatial distribution of weak and strong base-pairs among the duplex sequence. We show that the deviations from the two-state dehybridization mechanism are more pronounced when a strong core is flanked with weak extremities, while duplexes with a weak core but strong extremities exhibit a two-state behavior, which can be explained by the key role played by base fraying. This was later verified by generating artificial hairpin or circular states containing one or two locked duplex extremities, which results in an important reinforcement of the entire HB structure of the duplex and higher melting temperatures. Finally, we demonstrate that our results are little sensitive to the employed combination of RNA and water forcefields. The trends in thermal stability among the different sequences as well as the observed unfolding mechanisms (and the deviations from a two-state scenario) remain the same regardless of the employed atomistic models. However, our study points to possible limitations of recent reparametrizations of the Amber RNA forcefield, which sometimes results in duplexes that readily denature under ambient conditions, in contradiction with available experimental results.

Protein Folding Stability and Kinetics in Alginate Hydrogels.

Proteins are commonly encapsulated in alginate gels for drug delivery and tissue-engineering applications. However, there is limited knowledge of how encapsulation impacts intrinsic protein properties such as folding stability or unfolding kinetics. Here, we use fast relaxation imaging (FReI) to image protein unfolding in situ in alginate hydrogels after applying a temperature jump. Based on changes in the Förster resonance energy transfer (FRET) response of FRET-labeled phosphoglycerate kinase (PGK), we report the quantitative impact of multiple alginate hydrogel concentrations on protein stability and folding dynamics. The gels stabilize PGK by increasing its melting temperature up to 18.4 °C, and the stabilization follows a nonmonotonic dependence on the alginate density. In situ kinetic measurements also reveal that PGK deviates more from two-state folding behavior in denser gels and that the gel decreases the unfolding rate and accelerates the folding rate of PGK, compared to buffer. Phi-value analysis suggests that the folding transition state of an encapsulated protein is structurally similar to that of folded protein. This work reveals both beneficial and negative impacts of gel encapsulation on protein folding, as well as potential mechanisms contributing to altered stability.

Enhancement of Transient Current in MIS(p) Tunneling Diode with Reduced Reversed Bias Current by Oxide Removal at the Gate Edge

Transient behavior plays a critical role in electronic devices since it represents the potential to serve as memory applications. Recently, some transient current behavior of metal-insulator-semiconductor tunnel diode (MISTD) has been demonstrated with unconventional structures [1], [2]. However, due to the usage of multiple photomasks and the process complexity at the gate edge, these devices may have scale-down issues in advanced CMOS-compatible processes. In this work, a structure of edge-removed (ER) MISTD has been proposed. Compared to previous work [2], which removes surrounding oxide and Si substrate by trench forming, the ER MISTD improves the device performance by not damaging the Si substrate. The ER MISTD can remain the same transient current window (CW) while being operated at smaller write voltage and device area. With the advantages of a smaller device area, energy-efficient operations, and the simplicity of the fabrication process, the ER MISTD is promising to serve as dynamic memory applications.The schematics of the conventional co-planar and ER MISTD are shown in Fig. 1. With the removal of the surrounding gate oxide, ER MISTD demonstrates not only lower reverse bias current but also hysteresis displacement current in Fig. 2(a) compared to planar MISTD. The leakage current of ER MISTD is 3 orders of magnitude smaller than that of planar MISTD at VG = 2V. Fig. 2(b) shows the high-frequency capacitance-voltage measurement, which demonstrates enlarged deep depletion region of the ER MISTD. We further investigated the phenomenon by Sentaurus TCAD as shown in Fig. 3. The existence of oxide charges was also adopted in simulation. The deeper depletion phenomenon of ER MISTD can be attributed to the lack of minority carriers and oxide charges without the existence of surrounding gate oxide. Another evidence of the lack of minority carriers is shown in Fig. 4. The saturation current of ER MISTD remains lower while that of planar MISTD increases and eventually induces oxide breakdown around VG = 5V. It is also found that ER MISTD isn't in oxide breakdown when VG is over 40 V as shown in the inset of Fig. 4.Next, the enhanced two-state transient current behavior has been demonstrated in Fig. 5. The specific measurement steps are shown in the inset of Fig. 5(a) and 5(b). ER MISTD has better CW in both operations compared to planar MISTD. We further investigated the mechanism of the two operations.When VG is switched from 1V to 0V, the minority carriers (𝑄𝑖𝑛𝑣) would become excess carriers since no enough time for them to be recombined. Because ER MISTD has an insufficient supply of minority carriers due to the absence of gate edge fringing field, the electron tunnel current generated by excess carriers is smaller compared to planar MISTD. With a smaller electron tunnel current to compensate for the transient discharging current, the total transient read current of ER MISTD is larger than that of planar MISTD.When VG is switched from -0.5V to 0V, the depletion region is suddenly expanded and therefore generates extra displacement current. Since the change of depletion width of ER MISTD is larger, the positive displacement current is larger as well. Because the transient current is the summation of the positive discharging current and displacement current, the transient current of ER MISTD is larger.The endurance measurement of ER MISTD has been examined as shown in Fig. 6. Fig. 6(a) shows the endurance can be tested up to 5000 cycles between 1V and 0V without much degradation. Fig. 6(b) shows the endurance between 1V and -0.5V with 102 cycles.We further investigated the CW of two operations corresponding to oxide thickness. As shown in Fig. 7, when oxide becomes thicker, the transient behavior becomes larger due to the decrease of the tunneling probability. However, the CW would eventually decay when the oxide is too thick. This is because tunnel current will disappear at thicker oxide, the transient behavior is therefore reduced. The peak value of the CW is around EOT = 3.1 nm.In conclusion, ER MISTD shows enhanced transient current and reduced leakage current due to the enlarged deep depletion. We also proposed two-state memory operations and endurance measurement of ER MISTD. The correspondence of CW and oxide thickness has been investigated as well. Because of the above characteristics, ER MISTD shows its potential to serve as a dynamic memory application.Reference:[1] Huang S -W and Hwu J -G 2021 IEEE Trans. Electron Devices 68 6580-6585[2] Lin J -Y and Hwu J -G 2021 IEEE Trans. on Electron Devices 68 4189-4194 Figure 1

Two-State Lasing in Microdisk Laser Diodes with Quantum Dot Active Region

The two-state lasing phenomenon, which manifests itself in simultaneous laser emission through several optical transitions of quantum dots, is studied in microdisk diode lasers with different cavity diameters. The active region represents a multiply stacked array of self-organized InAs/InGaAs/GaAs quantum dots emitting in the wavelength range of 1.1–1.3 µm. Two-state lasing, which involves the ground-state and the first excited-state optical transitions, is observed in microdisks with cavity diameters of 20 to 28 µm, whereas two-state lasing via the first and the second excited-state optical transitions is observed in 9 µm microdisks. The threshold currents for one-state and two-state lasing are investigated as functions of the microdisk diameter. Optical loss in the microdisk lasers is evaluated by comparing the two-state lasing behavior of the microdisks with that of edge-emitting stripe lasers made of the same epitaxial wafer.

High Energy Channeling and Malleable Transition States: Molecular Dynamics Simulations and Free Energy Landscapes for the Thermal Unfolding of Protein U1A and 13 Mutants.

The spliceosome protein U1A is a prototype case of the RNA recognition motif (RRM) ubiquitous in biological systems. The in vitro kinetics of the chemical denaturation of U1A indicate that the unfolding of U1A is a two-state process but takes place via high energy channeling and a malleable transition state, an interesting variation of typical two-state behavior. Molecular dynamics (MD) simulations have been applied extensively to the study of two-state unfolding and folding of proteins and provide an opportunity to obtain a theoretical account of the experimental results and a molecular model for the transition state ensemble. We describe herein all-atom MD studies including explicit solvent of up to 100 ns on the thermal unfolding (UF) of U1A and 13 mutants. Multiple MD UF trajectories are carried out to ensure accuracy and reproducibility. A vector representation of the MD unfolding process in RMSD space is obtained and used to calculate a free energy landscape for U1A unfolding. A corresponding MD simulation and free energy landscape for the protein CI2, well known to follow a simple two state folding/unfolding model, is provided as a control. The results indicate that the unfolding pathway on the MD calculated free energy landscape of U1A shows a markedly extended transition state compared with that of CI2. The MD results support the interpretation of the observed chevron plots for U1A in terms of a high energy, channel-like transition state. Analysis of the MDUF structures shows that the transition state ensemble involves microstates with most of the RRM secondary structure intact but expanded by ~14% with respect to the radius of gyration. Comparison with results on a prototype system indicates that the transition state involves an ensemble of molten globule structures and extends over the region of ~1–35 ns in the trajectories. Additional MDUF simulations were carried out for 13 U1A mutants, and the calculated φ-values show close accord with observed results and serve to validate our methodology.

Competing interactions give rise to two-state behavior and switch-like transitions in charge-rich intrinsically disordered proteins.

The most commonly occurring intrinsically disordered proteins (IDPs) are polyampholytes, which are defined by the duality of low net charge per residue and high fractions of charged residues. Recent experiments have uncovered nuances regarding sequence–ensemble relationships of model polyampholytic IDPs. These include differences in conformational preferences for sequences with lysine vs. arginine and the suggestion that well-mixed sequences form a range of conformations, including globules, conformations with ensemble averages that are reminiscent of ideal chains, or self-avoiding walks. Here, we explain these observations by analyzing results from atomistic simulations. We find that polyampholytic IDPs generally sample two distinct stable states, namely, globules and self-avoiding walks. Globules are favored by electrostatic attractions between oppositely charged residues, whereas self-avoiding walks are favored by favorable free energies of hydration of charged residues. We find sequence-specific temperatures of bistability at which globules and self-avoiding walks can coexist. At these temperatures, ensemble averages over coexisting states give rise to statistics that resemble ideal chains without there being an actual counterbalancing of intrachain and chain-solvent interactions. At equivalent temperatures, arginine-rich sequences tilt the preference toward globular conformations whereas lysine-rich sequences tilt the preference toward self-avoiding walks. We also identify differences between aspartate- and glutamate-containing sequences, whereby the shorter aspartate side chain engenders preferences for metastable, necklace-like conformations. Finally, although segregation of oppositely charged residues within the linear sequence maintains the overall two-state behavior, compact states are highly favored by such systems.

Computational Modeling of the Thermodynamics of the Mesophilic and Thermophilic Mutants of Trp-Cage Miniprotein.

We characterize the folding–unfolding thermodynamics of two mutants of the miniprotein Trp-cage by combining extended molecular dynamics simulations and an advanced statistical–mechanical-based approach. From a set of molecular dynamics simulations in an explicit solvent performed along a reference isobar, we evaluated the structural and thermodynamic behaviors of a mesophilic and a thermophilic mutant of the Trp-cage and their temperature dependence. In the case of the thermophilic mutant, computational data confirm that our theoretical–computational approach is able to reproduce the available experimental estimate with rather good accuracy. On the other hand, the mesophilic mutant does not show a clear two-state (folded and unfolded) behavior, preventing us from reconstructing its thermodynamics; thus, an analysis of its structural behavior along a reference isobar is presented. Our results show that an extended sampling of these kinds of systems coupled to an advanced statistical–mechanical-based treatment of the data can provide an accurate description of the folding–unfolding thermodynamics along a reference isobar, rationalizing the discrepancies between the simulated and experimental systems.

Tunable Planar Hall Effect in (Ga,Mn)(Bi,As) Epitaxial Layers.

We have thoroughly investigated the planar Hall effect (PHE) in the epitaxial layers of the quaternary compound (Ga,Mn)(Bi,As). The addition of a small amount of heavy Bi atoms to the prototype dilute ferromagnetic semiconductor (Ga,Mn)As enhances significantly the spin–orbit coupling strength in its valence band, which essentially modifies certain magnetoelectric properties of the material. Our investigations demonstrate that an addition of just 1% Bi atomic fraction, substituting As atoms in the (Ga,Mn)As crystal lattice, causes an increase in the PHE magnitude by a factor of 2.5. Moreover, Bi incorporation into the layers strongly enhances their coercive fields and uniaxial magneto-crystalline anisotropy between the in-plane 〈110〉 crystallographic directions in the layers grown under a compressive misfit strain. The displayed two-state behaviour of the PHE resistivity at zero magnetic field, which may be tuned by the control of applied field orientation, could be useful for application in spintronic devices, such as nonvolatile memory elements.

The Horseshoe Abyssal plain Thrust could be the source of the 1755 Lisbon earthquake and tsunami

The southwest Iberia margin is widely believed to have hosted the 1755 Great Lisbon earthquake and ensuing tsunami, one of the most destructive natural events in European history. Here we combine geophysical data and numerical tsunami modelling to investigate the source and mechanism responsible for this event. We find that an intra-plate, lithospheric¬-scale thrust fault located at the Horseshoe Abyssal Plain coincides with the location and focal mechanisms of the largest regional earthquakes and is likely to have suitable dimensions and fault-rock properties to account for the magnitude of the 1755 event. We present tsunami simulations with this fault as the source, and find that they reproduce reported tsunami energy propagation patterns, arrival-times and run up heights more successfully than other modelled sources. We propose that a reverse dip-slip mechanism on the northwest verging Horseshoe Abyssal plain Thrust, combined with the two-state mechanical behaviour of serpentinite, is the most likely candidate for the source of the 1755 Great Lisbon earthquake and for other recent large regional earthquakes.

Presynaptic Gαo (GOA-1) signals to depress command neuron excitability and allow stretch-dependent modulation of egg laying in Caenorhabditis elegans.

Egg laying in the nematode worm Caenorhabditis elegans is a two-state behavior modulated by internal and external sensory input. We have previously shown that homeostatic feedback of embryo accumulation in the uterus regulates bursting activity of the serotonergic HSN command neurons that sustains the egg-laying active state. How sensory feedback of egg release signals to terminate the egg-laying active state is less understood. We find that Gαo, a conserved Pertussis Toxin-sensitive G protein, signals within HSN to inhibit egg-laying circuit activity and prevent entry into the active state. Gαo signaling hyperpolarizes HSN, reducing HSN Ca2+ activity and input onto the postsynaptic vulval muscles. Loss of inhibitory Gαo signaling uncouples presynaptic HSN activity from a postsynaptic, stretch-dependent homeostat, causing precocious entry into the egg-laying active state when only a few eggs are present in the uterus. Feedback of vulval opening and egg release activates the uv1 neuroendocrine cells which release NLP-7 neuropeptides which signal to inhibit egg laying through Gαo-independent mechanisms in the HSNs and Gαo-dependent mechanisms in cells other than the HSNs. Thus, neuropeptide and inhibitory Gαo signaling maintain a bi-stable state of electrical excitability that dynamically controls circuit activity in response to both external and internal sensory input to drive a two-state behavior output.

Conformational and migrational dynamics of slipped-strand DNA three-way junctions containing trinucleotide repeats

Expansions of CAG/CTG trinucleotide repeats in DNA are the cause of at least 17 degenerative human disorders, including Huntington’s Disease. Repeat instability is thought to occur via the formation of intrastrand hairpins during replication, repair, recombination, and transcription though relatively little is known about their structure and dynamics. We use single-molecule Förster resonance energy transfer to study DNA three-way junctions (3WJs) containing slip-outs composed of CAG or CTG repeats. 3WJs that only have repeats in the slip-out show two-state behavior, which we attribute to conformational flexibility at the 3WJ branchpoint. When the triplet repeats extend into the adjacent duplex, additional dynamics are observed, which we assign to interconversion of positional isomers. We propose a branchpoint migration model that involves conformational rearrangement, strand exchange, and bulge-loop movement. This migration has implications for how repeat slip-outs are processed by the cellular machinery, disease progression, and their development as drug targets.

Time-dependent communication between multiple amino acids during protein folding

Cooperativity is considered to be a key organizing principle behind biomolecular assembly, recognition and folding. However, it has remained very challenging to quantitatively characterize how cooperative processes occur on a concerted, multiple-interaction basis. Here, we address how and when the folding process is cooperative on a molecular scale. To this end, we analyze multipoint time-correlation functions probing time-dependent communication between multiple amino acids, which were computed from long folding simulation trajectories. We find that the simultaneous multiple amino-acid contact formation, which is absent in the unfolded state, starts to develop only upon entering the folding transition path. Interestingly, the transition state, whose presence is connected to the macrostate cooperative behavior known as the two-state folding, can be identified as the state in which the amino-acid cooperativity is maximal. Thus, our work not only provides a new mechanistic view on how protein folding proceeds on a multiple-interaction basis, but also offers a conceptually novel characterization of the folding transition state and the molecular origin of the phenomenological cooperative folding behavior. Moreover, the multipoint correlation function approach adopted here is general and can be used to expand the understanding of cooperative processes in complex chemical and biomolecular systems.

Double-Well Ultra-Coarse-Grained Model to Describe Protein Conformational Transitions.

The double-well model is usually used to describe the conformational transition between two states of a protein. Since conformational changes usually occur within a relatively large time scale, coarse-grained models are often used to accelerate the dynamic process due to their inexpensive computational cost. In this work, we develop a double-well ultra-coarse-grained (DW-UCG) model to describe the conformational transitions of the adenylate kinase, glutamine-binding protein, and lactoferrin. The coarse-grained simulation results show that the DW-UCG model of adenylate kinase captures the crucial intermediate states in the LID-closing and NMP-closing pathways, reflecting the key secondary structural changes in the conformational transition. A comparison of the different DW-UCG models of adenylate kinase indicates that an appropriate choice of bead resolution could generate the free energy landscape that is comparable to that from the residue-based model. The coarse-grained simulations for the glutamine-binding protein and lactoferrin also demonstrate that the DW-UCG model is valid in reproducing the correct two-state behavior for their functional study, which indicates the potential application of the DW-UCG model in investigating the mechanism of conformational changes of large proteins.

Efficient and exact sampling of transition path ensembles on Markovian networks.

The problem of flickering trajectories in standard kinetic Monte Carlo (kMC) simulations prohibits sampling of the transition path ensembles (TPEs) on Markovian networks representing many slow dynamical processes of interest. In the present contribution, we overcome this problem using knowledge of the metastable macrostates, determined by an unsupervised community detection algorithm, to perform enhanced sampling kMC simulations. We implement two accelerated kMC methods to simulate the nonequilibrium stochastic dynamics on arbitrary Markovian networks, namely, weighted ensemble (WE) sampling and kinetic path sampling (kPS). WE-kMC utilizes resampling in pathway space to maintain an ensemble of representative trajectories covering the state space, and kPS utilizes graph transformation to simplify the description of an escape trajectory from a trapping energy basin. Both methods sample individual trajectories governed by the linear master equation with the correct statistical frequency. We demonstrate that they allow for efficient estimation of the time-dependent occupation probability distributions for the metastable macrostates, and of TPE statistics, such as committor functions and first passage time distributions. kPS is particularly attractive, since its efficiency is essentially independent of the degree of metastability, and we suggest how the algorithm could be coupled with other enhanced sampling methodologies. We illustrate our approach with results for a network representing the folding transition of a tryptophan zipper peptide, which exhibits a separation of characteristic timescales. We highlight some salient features of the dynamics, most notably, strong deviations from two-state behavior, and the existence of multiple competing mechanisms.

Life under Extreme Conditions: Aeropyrum pernix and Pernisine

Life under Extreme Conditions: Aeropyrum pernix and Pernisine

Complex magnetic ordering in nanoporous [Co/Pd]5-IrMn multilayers with perpendicular magnetic anisotropy and its impact on magnetization reversal and magnetoresistance.

We have systematically investigated the magnetization reversal characteristics and magnetoresistance of continuous and nanoporous [Co/Pd]5-IrMn multilayered thin films with perpendicular magnetic anisotropy at different temperatures (4-300 K). For their nanostructuring, porosity was induced by means of deposition onto templates of anodized titania with small (∼30 nm in diameter) homogeneously distributed pores. The magnetization reversal and magnetoresistance of the porous films were found to be closely related to the splitting of the ferromagnetic material into regions with different magnetic properties, in correlation with the complex morphology of the porous system. Independent magnetization reversal is detected for these regions, and is accompanied by its strong impact on the magnetic order in the capping IrMn layer. Electron-magnon scattering is found to be a dominant mechanism of magnetoresistance, determining its almost linear field dependence in a high magnetic field and contributing to its magnetoresistance behavior, similar to magnetization reversal, in a low magnetic field. Partial rotation of IrMn magnetic moments, consistent with the magnetization reversal of the ferromagnet, is proposed as an explanation for the two-state resistance behavior observed in switching between high-resistive and low-resistive values at the magnetization reversal of the porous system studied.

Mapping Multiple Distances in a Multidomain Protein for the Identification of Folding Intermediates

The investigation and understanding of the folding mechanism of multidomain proteins is still a challenge in structural biology. The use of single-molecule Förster resonance energy transfer offers a unique tool to map conformational changes within the protein structure. Here, we present a study following denaturant-induced unfolding transitions of yeast phosphoglycerate kinase by mapping several inter- and intradomain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. On the one hand, the development of the interdomain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intradomain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intradomain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by single-molecule Förster resonance energy transfer measurements. Our results suggest that, to fully characterize the complex folding and unfolding mechanism of multidomain proteins, it is necessary to monitor multiple intra- and interdomain distances because a single reporter can lead to a misleading, partial, or oversimplified interpretation.

Antibody-induced crosslinking and cholesterol-sensitive, anomalous diffusion of nicotinic acetylcholine receptors.

Synaptic strength depends on the number of cell-surface neurotransmitter receptors in dynamic equilibrium with intracellular pools. Dysregulation of this homeostatic balance occurs, for example in myasthenia gravis, an autoimmune disease characterized by a decrease in the number of postsynaptic nicotinic acetylcholine receptors (nAChRs). Monoclonal antibody mAb35 mimics this effect. Here we use STORM nanoscopy to characterize the individual and ensemble dynamics of monoclonal antibody-crosslinked receptors in the clonal cell line CHO-K1/A5, which robustly expresses adult muscle-type nAChRs. Antibody labeling of live cells results in 80% receptor immobilization. The remaining mobile fraction exhibits a heterogeneous combination of Brownian and anomalous diffusion. Single-molecule trajectories exhibit a two-state switching behavior between free Brownian walks and anticorrelated walks within confinement areas. The latter act as permeable fences (~34nm radius, ~400ms lifetime). Dynamic clustering, trapping, and immobilization also occur in larger nanocluster zones (120-180nm radius) with longer lifetimes (11±1s), in a strongly cholesterol-sensitive manner. Cholesterol depletion increases the size of the clustering phenomenon; cholesterol enrichment has the opposite effect. The disclosed high proportion of monoclonal antibody-crosslinked immobile receptors, together with their anomalous, cholesterol-sensitive diffusion and clustering, provides new insights into the antibody-enhanced antigenic modulation that leads to physiopathological internalization and degradation of receptors in myasthenia.